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PPP2CA Is a Novel Therapeutic Target in Neuroblastoma Cells That Can Be Activated by the SET Inhibitor OP449.

In Front Oncol on 12 July 2022 by Galiger, C., Dahlhaus, M., et al.

Fig.5.A

OP449 reactivates PP2A in neuroblastoma (NB) cells. (A) OP449 dephosphorylates PPP2CA at Y307. SH-SY5Y and KELLY NB cells were treated for 4 h with increasing concentrations of OP449. Western blotting for pY307 of PPP2CA and for PPP2CA is shown. β-Actin was used as loading control. Full-length We... more

OP449 reactivates PP2A in neuroblastoma (NB) cells. (A) OP449 dephosphorylates PPP2CA at Y307. SH-SY5Y and KELLY NB cells were treated for 4 h with increasing concentrations of OP449. Western blotting for pY307 of PPP2CA and for PPP2CA is shown. β-Actin was used as loading control. Full-length Western blotting is shown in Supplementary Figure 2B. (B) OP449 reactivates PP2A. Cells were treated for 4 h with increasing concentrations of OP449 and lysed. PPP2CA was immuno-precipitated, and PP2A activity was determined by colorimetric assay. Experiments were repeated three times. Data are expressed as means ± SEM of triplicates and are analyzed by one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. less

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ROCK1 activation-mediated mitochondrial translocation of Drp1 and cofilin are required for arnidiol-induced mitochondrial fission and apoptosis.

In J Exp Clin Cancer Res on 19 February 2020 by Hu, J., Zhang, H., et al.

Fig.7.A

ROCK1 activation is involved in arnidiol-mediated dephosphorylation and mitochondrial translocation of Drp1 and cofilin, mitochondrial fission and apoptosis. a MDA-MB-231 cells were treated with various concentrations of Arn for 48 h or with Arn (60 μM) for different time intervals as indicated, ... more

ROCK1 activation is involved in arnidiol-mediated dephosphorylation and mitochondrial translocation of Drp1 and cofilin, mitochondrial fission and apoptosis. a MDA-MB-231 cells were treated with various concentrations of Arn for 48 h or with Arn (60 μM) for different time intervals as indicated, WCL were prepared and subjected to western blot using antibodies against PP1, PP2A and ROCK1. GAPDH was used as loading control. b MCF-7, Eca109, SMMC-7721 and A549 cells were treated with Arn (60 μM) for 48 h, WCL were prepared and subjected to Western blot analysis using antibodies against PP1, PP2A and ROCK1. GAPDH was used as loading control. For c-i, cells stably expressing shControl or shROCK1 were treated with Arn (60 μM) for 48 h. c and d WCL, cytosol and mitochondrial fractions were prepared and subjected to western blot using antibodies against ROCK1, PP1, PP2A, p-Drp1 (S637), p-Cofilin, Drp1 and Cofilin, GAPDH and COX IV were used as loading controls. e The colocalization of Cofilin (red), Drp1 (green), and MitoTracker (blue) was examined using confocal microscopy. Scale bars: 10 μm. f Mitochondrial morphology was observed by MitoTracker Red CMXRos staining and confocal microscopy. Scale bars: 10 μm. g Mitochondrial length was measured with ImageJ software. 50 cells of 3 independent experiments (mean ± SD, ***P < 0.001). h Apoptosis was detected by flow cytometry analysis (mean ± SD for 3 separate experiments, ***P < 0.001). i WCL, cytosol fractions were prepared and subjected to western blot using antibodies against total PRAP, C-PARP, C-Caspase-3 and Cyto C. GAPDH was used as loading control less

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Protein phosphatase 2A interacts with Verthandi/Rad21 to regulate mitosis and organ development in Drosophila.

In Sci Rep on 20 May 2019 by Kim, L. H., Hong, S. T., et al.

Fig.5.A

mts knockdown causes reduction of Vtd level in S2 cells and wing imaginal disc. (a) Immunoblot using extracts from S2 cells treated with control dsRNA (pSK vector) or mts dsRNA. Mts level is significantly reduced from Day 4. Vtd level is also reduced from Day 4. Immunoblot with anti-Tubulin and a... more

mts knockdown causes reduction of Vtd level in S2 cells and wing imaginal disc. (a) Immunoblot using extracts from S2 cells treated with control dsRNA (pSK vector) or mts dsRNA. Mts level is significantly reduced from Day 4. Vtd level is also reduced from Day 4. Immunoblot with anti-Tubulin and anti-Histone H3 staining is used as controls to compare the amount of Mts or Vtd. (b–b”’,c–c”’) Wing imaginal discs were stained by DAPI (blue), anti-Vtd (red) antibodies. (b–b”’) GFP+ cells are mts knockdown clones induced by hs-FLP; Ay > mts RNAi. mts RNAi clones show reduction of Vtd. Scale bar, 20 µm. (c–c”’) tws60 mutant clones marked by the lack of GFP expression show reduction of Vtd staining. Scale bar, 20 µm. (d) Immunoblot using extract from w1118 wing imaginal disc (control) or tws60 homozygous wing imaginal disc (left panel). Quantitative analysis (right panel). Vtd levels are normalized to the amount of Histone H3. Error bars are s.d. n = 3. **P < 0.01 (t-test). (a,d) Full-length blots are presented in Supplementary Fig. 6. less

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Phospholipase C-related catalytically inactive protein (PRIP) regulates lipolysis in adipose tissue by modulating the phosphorylation of hormone-sensitive lipase.

In PLoS One on 20 June 2014 by Okumura, T., Harada, K., et al.

Fig.4.A

PP2A and PP1 are translocated from the cytosol to lipid droplets in adipocytes in response to adrenaline stimulation.(A–C) Translocation of PP2A (A, B) and PP1 (A, C) to the lipid droplet fraction in response to stimulation with 1 µM adrenaline (Adrn). The bar graph shows the amount of PP2A (B) a... more

PP2A and PP1 are translocated from the cytosol to lipid droplets in adipocytes in response to adrenaline stimulation.(A–C) Translocation of PP2A (A, B) and PP1 (A, C) to the lipid droplet fraction in response to stimulation with 1 µM adrenaline (Adrn). The bar graph shows the amount of PP2A (B) and PP1 (C) in fat (black) and sup (white) fractions, respectively. A typical image from four independent experiments is shown (A). (D, E) Phosphatase activity in floating fat-cake fraction. Phosphatase activities of PP2A (D, n = 4) and PP1 (E, n = 5) were measured using floating fat-cake fractions from WT and PRIP-DKO explants treated with or without 1 µM adrenaline. The data represent mean ±SEM; *P<0.05, **P<0.01, ***P<0.001 and n.s. (not significant) versus the corresponding values for paired bars connected by solid and dotted lines (B–E). less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.4.H

NAP1 interacts biochemically with the core cohesin complex and PP2A.(A) Proteomic analysis of the NAP1 interaction network. NAP1 and associated proteins were immunopurified from 0–12 hour Drosophila embryo nuclear extracts (NE) using affinity-purified antibodies raised against NAP1. After extensi... more

NAP1 interacts biochemically with the core cohesin complex and PP2A.(A) Proteomic analysis of the NAP1 interaction network. NAP1 and associated proteins were immunopurified from 0–12 hour Drosophila embryo nuclear extracts (NE) using affinity-purified antibodies raised against NAP1. After extensive washes with a buffer containing 600 mM KCl and 0.1% NP40, bound proteins were resolved by SDS-PAGE, visualized by coomassie staining and identified by mass-spectrometry. NAP1, RLAF subunits (SIN3A, LID, EMSY, PF1, RPD3 and MRG15) and cohesin subunits (SMC1/3, SA and RAD21) are indicated. A comprehensive list of identified proteins is provided in Table S1. (B) Identification of SA-associated factors. For a complete list of associated factors see Table S1. (C) Identification of SMC1-associated factors (see Table S1). (D) Identification of PP2A interaction network. Purification of PP2A-associated factors was performed as described above with antibodies against the catalytic subunit. For a list of selected factors associated with PP2A see Table S2. Protein bands corresponding to PP2A catalytic subunit, NAP1 and cohesin subunits, identified by mass spectrometric analysis are indicted. (E) SA was IPed from NE, followed by extensive washes with a buffer containing 600 mM KCl and 0.1% NP40. The binding of NAP1, cohesin subunits, PP2A, MeiS332, and histone chaperones ASF1 and CAF1 was assayed by immunoblotting using the appropriate antibodies. Mock IPs were performed with pre-immune serum. Input represents 10% of the binding reactions. A lack of SA association with MeiS332 was also confirmed under lower stringency (200 mM KCl, 0.1% NP40). (F) Co-IP analysis of NAP1 interactions. Analysis as described above. (G) Co-IP analysis of PP2A interactions. All PP2A interactions were detected under high stringency (600 mM KCl), except for MeiS332, which can be detected only at lower stringency (200 mM KCl). (H) Sephacryl S-300 size-exclusion chromatography analysis of NAP1, cohesin, PP2A and MeiS332. The indicated fractions were resolved by SDS-PAGE followed by immunoblotting. NAP1, PP2A and cohesin subunits SA, SMC1 and RAD21 co-eluted in column fractions corresponding to an apparent molecular mass of ∼1.5 MDa. In addition, NAP1 and SA, but not SMC1 or RAD21, eluted in lower molecular weight fractions of ∼300 kDa. Voided volume (void), determined by Blue Dextran 2000, and elution of the markers ferritin (440 kDa) and aldolase (158 kDa) are indicated. (I) co-IPs of NAP1 and SA from pooled high molecular weight S-300 column fractions (#12–14). IPs were performed as above. Input represents 10% of the binding reactions. (J) co-IPs of NAP1 and SA from pooled lower molecular weight fractions (#27–29). As S-300 fractions #27–29 lack SMC1 and RAD21, NE was used as a control for Western blotting efficiency. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.4.E

NAP1 interacts biochemically with the core cohesin complex and PP2A.(A) Proteomic analysis of the NAP1 interaction network. NAP1 and associated proteins were immunopurified from 0–12 hour Drosophila embryo nuclear extracts (NE) using affinity-purified antibodies raised against NAP1. After extensi... more

NAP1 interacts biochemically with the core cohesin complex and PP2A.(A) Proteomic analysis of the NAP1 interaction network. NAP1 and associated proteins were immunopurified from 0–12 hour Drosophila embryo nuclear extracts (NE) using affinity-purified antibodies raised against NAP1. After extensive washes with a buffer containing 600 mM KCl and 0.1% NP40, bound proteins were resolved by SDS-PAGE, visualized by coomassie staining and identified by mass-spectrometry. NAP1, RLAF subunits (SIN3A, LID, EMSY, PF1, RPD3 and MRG15) and cohesin subunits (SMC1/3, SA and RAD21) are indicated. A comprehensive list of identified proteins is provided in Table S1. (B) Identification of SA-associated factors. For a complete list of associated factors see Table S1. (C) Identification of SMC1-associated factors (see Table S1). (D) Identification of PP2A interaction network. Purification of PP2A-associated factors was performed as described above with antibodies against the catalytic subunit. For a list of selected factors associated with PP2A see Table S2. Protein bands corresponding to PP2A catalytic subunit, NAP1 and cohesin subunits, identified by mass spectrometric analysis are indicted. (E) SA was IPed from NE, followed by extensive washes with a buffer containing 600 mM KCl and 0.1% NP40. The binding of NAP1, cohesin subunits, PP2A, MeiS332, and histone chaperones ASF1 and CAF1 was assayed by immunoblotting using the appropriate antibodies. Mock IPs were performed with pre-immune serum. Input represents 10% of the binding reactions. A lack of SA association with MeiS332 was also confirmed under lower stringency (200 mM KCl, 0.1% NP40). (F) Co-IP analysis of NAP1 interactions. Analysis as described above. (G) Co-IP analysis of PP2A interactions. All PP2A interactions were detected under high stringency (600 mM KCl), except for MeiS332, which can be detected only at lower stringency (200 mM KCl). (H) Sephacryl S-300 size-exclusion chromatography analysis of NAP1, cohesin, PP2A and MeiS332. The indicated fractions were resolved by SDS-PAGE followed by immunoblotting. NAP1, PP2A and cohesin subunits SA, SMC1 and RAD21 co-eluted in column fractions corresponding to an apparent molecular mass of ∼1.5 MDa. In addition, NAP1 and SA, but not SMC1 or RAD21, eluted in lower molecular weight fractions of ∼300 kDa. Voided volume (void), determined by Blue Dextran 2000, and elution of the markers ferritin (440 kDa) and aldolase (158 kDa) are indicated. (I) co-IPs of NAP1 and SA from pooled high molecular weight S-300 column fractions (#12–14). IPs were performed as above. Input represents 10% of the binding reactions. (J) co-IPs of NAP1 and SA from pooled lower molecular weight fractions (#27–29). As S-300 fractions #27–29 lack SMC1 and RAD21, NE was used as a control for Western blotting efficiency. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.4.D

NAP1 interacts biochemically with the core cohesin complex and PP2A.(A) Proteomic analysis of the NAP1 interaction network. NAP1 and associated proteins were immunopurified from 0–12 hour Drosophila embryo nuclear extracts (NE) using affinity-purified antibodies raised against NAP1. After extensi... more

NAP1 interacts biochemically with the core cohesin complex and PP2A.(A) Proteomic analysis of the NAP1 interaction network. NAP1 and associated proteins were immunopurified from 0–12 hour Drosophila embryo nuclear extracts (NE) using affinity-purified antibodies raised against NAP1. After extensive washes with a buffer containing 600 mM KCl and 0.1% NP40, bound proteins were resolved by SDS-PAGE, visualized by coomassie staining and identified by mass-spectrometry. NAP1, RLAF subunits (SIN3A, LID, EMSY, PF1, RPD3 and MRG15) and cohesin subunits (SMC1/3, SA and RAD21) are indicated. A comprehensive list of identified proteins is provided in Table S1. (B) Identification of SA-associated factors. For a complete list of associated factors see Table S1. (C) Identification of SMC1-associated factors (see Table S1). (D) Identification of PP2A interaction network. Purification of PP2A-associated factors was performed as described above with antibodies against the catalytic subunit. For a list of selected factors associated with PP2A see Table S2. Protein bands corresponding to PP2A catalytic subunit, NAP1 and cohesin subunits, identified by mass spectrometric analysis are indicted. (E) SA was IPed from NE, followed by extensive washes with a buffer containing 600 mM KCl and 0.1% NP40. The binding of NAP1, cohesin subunits, PP2A, MeiS332, and histone chaperones ASF1 and CAF1 was assayed by immunoblotting using the appropriate antibodies. Mock IPs were performed with pre-immune serum. Input represents 10% of the binding reactions. A lack of SA association with MeiS332 was also confirmed under lower stringency (200 mM KCl, 0.1% NP40). (F) Co-IP analysis of NAP1 interactions. Analysis as described above. (G) Co-IP analysis of PP2A interactions. All PP2A interactions were detected under high stringency (600 mM KCl), except for MeiS332, which can be detected only at lower stringency (200 mM KCl). (H) Sephacryl S-300 size-exclusion chromatography analysis of NAP1, cohesin, PP2A and MeiS332. The indicated fractions were resolved by SDS-PAGE followed by immunoblotting. NAP1, PP2A and cohesin subunits SA, SMC1 and RAD21 co-eluted in column fractions corresponding to an apparent molecular mass of ∼1.5 MDa. In addition, NAP1 and SA, but not SMC1 or RAD21, eluted in lower molecular weight fractions of ∼300 kDa. Voided volume (void), determined by Blue Dextran 2000, and elution of the markers ferritin (440 kDa) and aldolase (158 kDa) are indicated. (I) co-IPs of NAP1 and SA from pooled high molecular weight S-300 column fractions (#12–14). IPs were performed as above. Input represents 10% of the binding reactions. (J) co-IPs of NAP1 and SA from pooled lower molecular weight fractions (#27–29). As S-300 fractions #27–29 lack SMC1 and RAD21, NE was used as a control for Western blotting efficiency. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.5.B

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immun... more

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (B) or SMC1 (C) as described in Figure 4B–C. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (D) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (E) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (D), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (Figure S5A). (F) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (G) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (D). Similar results were obtained for SMC1 IPs from colhicine-treated cells (Figure S6). (H) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (I) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.5.G

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immun... more

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (B) or SMC1 (C) as described in Figure 4B–C. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (D) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (E) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (D), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (Figure S5A). (F) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (G) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (D). Similar results were obtained for SMC1 IPs from colhicine-treated cells (Figure S6). (H) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (I) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.5.D

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immun... more

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (B) or SMC1 (C) as described in Figure 4B–C. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (D) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (E) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (D), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (Figure S5A). (F) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (G) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (D). Similar results were obtained for SMC1 IPs from colhicine-treated cells (Figure S6). (H) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (I) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.5.C

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immun... more

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (B) or SMC1 (C) as described in Figure 4B–C. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (D) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (E) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (D), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (Figure S5A). (F) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (G) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (D). Similar results were obtained for SMC1 IPs from colhicine-treated cells (Figure S6). (H) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (I) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion. less

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Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

In PLoS Genet on 3 October 2013 by Moshkin, Y. M., Doyen, C. M., et al.

Fig.5.H

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immun... more

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.(A) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (B–C) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (B) or SMC1 (C) as described in Figure 4B–C. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (D) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (E) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (D), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (Figure S5A). (F) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (G) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (D). Similar results were obtained for SMC1 IPs from colhicine-treated cells (Figure S6). (H) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (I) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion. less

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