pp1 γ Search Results


anti pp1γ  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology anti pp1γ
Anti Pp1γ, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/PP1%CE%B3+Antibody/pm26542020-145-2-9
Average 93 stars, based on 1 article reviews
anti pp1γ - by Bioz Stars, 2026-09
93/100 stars
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pp1γ sirna  (Santa Cruz Biotechnology)
92
Santa Cruz Biotechnology pp1γ sirna
Pp1γ Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/PP1%CE%B3+siRNA/pmc01920181-512-4-7
Average 92 stars, based on 1 article reviews
pp1γ sirna - by Bioz Stars, 2026-09
92/100 stars
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pp1γ  (Santa Cruz Biotechnology)
92
Santa Cruz Biotechnology pp1γ
NIPP1 inhibited <t>PP1γ</t> activity toward H3‐pThr 11 dephosphorylation. A, Venn diagram showing the number of PIPs that overlap among nucleus (GO:0 005 634), transcription, DNA‐templated (GO:0 006 351) and regulators of catalytic activity. Regulators of catalytic activity contain positive regulation of catalytic activity (GO:0 043 085), negative regulation of catalytic activity (GO:0 043 086), negative regulation of phosphatase activity (GO:0 010 923) and positive regulation of phosphoprotein phosphatase activity (GO:0 032 516). B, HCT116 cells were treated with UV and harvested at the indicated times. The chromatin fraction, soluble fraction, and whole cell extract (WCE) were subjected to immunoblotting using the indicated antibodies. IKKα and H3 were used as markers of soluble protein or chromatin protein, respectively. Relative band intensity of NIPP1 (WCE) was normalized by H3, compared with 0 h. Relative band intensity of NIPP1 on chromatin and soluble fractions was normalized by NIPP1 (WCE). Signals were quantified using ImageJ software. C, HCT116 cells were transfected with empty vector or vectors expressing myc‐his‐PNUTS. After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. D, Domain structure of NIPP1‐RATA and NIPP1‐ΔC mutant are shown. NIPP1 contains PP1‐binding region harboring the RVTF sequence (top). HCT116 cells were transfected with empty vector or vectors expressing the indicated NIPP1 (bottom). After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. Signals were quantified using Image Lab. E, HCT116 cells were transfected with either control, PNUTS, or NIPP1 siRNAs. After 70 h, the cells were treated with (+) or without (−) UV. Chromatin fractions were prepared for immunoblotting after 2 h incubation. Signals were quantified using Image Lab. F, In vitro phosphatase assay was performed using purified PP1γ and NIPP1. PP1γ‐WT, or T311A preincubated with his‐NIPP1 were incubated with chromatin. The phosphorylation of H3‐Thr 11 was monitored by immunoblotting using H3‐pThr 11 antibodies. The relative band intensity of H3‐pThr 11 was normalized by H3, compared with the control, and quantified using ImageJ software. G, HCT116 cells were treated with 25 J/m 2 UV and harvested at the indicated times (left). The chromatin fraction was subjected to immunoblotting using the indicated antibodies. H3 was used as markers of chromatin protein. Relative band intensity of NIPP1 was normalized by H3, compared with 0 h. Signals were quantified using Image Lab. Real‐time PCR analysis of CDK1 and CCNB1 expression in HCT116 cells (right). Data are provided as the mean ± SEM of 3 independent experiments. The results were considered statistically significant at * P < .05 and ** P < .01 [Correction added on 14 May 2021, after first online publication: Figure 1D has been corrected.]
Pp1γ, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/PP1%CE%B3+shRNA+(h)+Lentiviral+Particles/pmc08253265-93-101-103
Average 92 stars, based on 1 article reviews
pp1γ - by Bioz Stars, 2026-09
92/100 stars
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rabbit anti-pp1γ (gtx105618)  (GeneTex)
90
GeneTex rabbit anti-pp1γ (gtx105618)
NIPP1 inhibited <t>PP1γ</t> activity toward H3‐pThr 11 dephosphorylation. A, Venn diagram showing the number of PIPs that overlap among nucleus (GO:0 005 634), transcription, DNA‐templated (GO:0 006 351) and regulators of catalytic activity. Regulators of catalytic activity contain positive regulation of catalytic activity (GO:0 043 085), negative regulation of catalytic activity (GO:0 043 086), negative regulation of phosphatase activity (GO:0 010 923) and positive regulation of phosphoprotein phosphatase activity (GO:0 032 516). B, HCT116 cells were treated with UV and harvested at the indicated times. The chromatin fraction, soluble fraction, and whole cell extract (WCE) were subjected to immunoblotting using the indicated antibodies. IKKα and H3 were used as markers of soluble protein or chromatin protein, respectively. Relative band intensity of NIPP1 (WCE) was normalized by H3, compared with 0 h. Relative band intensity of NIPP1 on chromatin and soluble fractions was normalized by NIPP1 (WCE). Signals were quantified using ImageJ software. C, HCT116 cells were transfected with empty vector or vectors expressing myc‐his‐PNUTS. After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. D, Domain structure of NIPP1‐RATA and NIPP1‐ΔC mutant are shown. NIPP1 contains PP1‐binding region harboring the RVTF sequence (top). HCT116 cells were transfected with empty vector or vectors expressing the indicated NIPP1 (bottom). After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. Signals were quantified using Image Lab. E, HCT116 cells were transfected with either control, PNUTS, or NIPP1 siRNAs. After 70 h, the cells were treated with (+) or without (−) UV. Chromatin fractions were prepared for immunoblotting after 2 h incubation. Signals were quantified using Image Lab. F, In vitro phosphatase assay was performed using purified PP1γ and NIPP1. PP1γ‐WT, or T311A preincubated with his‐NIPP1 were incubated with chromatin. The phosphorylation of H3‐Thr 11 was monitored by immunoblotting using H3‐pThr 11 antibodies. The relative band intensity of H3‐pThr 11 was normalized by H3, compared with the control, and quantified using ImageJ software. G, HCT116 cells were treated with 25 J/m 2 UV and harvested at the indicated times (left). The chromatin fraction was subjected to immunoblotting using the indicated antibodies. H3 was used as markers of chromatin protein. Relative band intensity of NIPP1 was normalized by H3, compared with 0 h. Signals were quantified using Image Lab. Real‐time PCR analysis of CDK1 and CCNB1 expression in HCT116 cells (right). Data are provided as the mean ± SEM of 3 independent experiments. The results were considered statistically significant at * P < .05 and ** P < .01 [Correction added on 14 May 2021, after first online publication: Figure 1D has been corrected.]
Rabbit Anti Pp1γ (Gtx105618), supplied by GeneTex, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/rabbit+anti+pp1%CE%B3++gtx105618+/10__1091_slash_mbc__e11___01___0012-216-64-67
Average 90 stars, based on 1 article reviews
rabbit anti-pp1γ (gtx105618) - by Bioz Stars, 2026-09
90/100 stars
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pp1γ  (SignaGen)
90
SignaGen pp1γ
Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, <t>PP1γ,</t> and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.
Pp1γ, supplied by SignaGen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/pp1%CE%B3/pmc06422231-54-33-35
Average 90 stars, based on 1 article reviews
pp1γ - by Bioz Stars, 2026-09
90/100 stars
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hela flp-in pp1γ−2xstrep cell line  (Hemmo Pharmaceuticals)
90
Hemmo Pharmaceuticals hela flp-in pp1γ−2xstrep cell line
Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, <t>PP1γ,</t> and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.
Hela Flp In Pp1γ−2xstrep Cell Line, supplied by Hemmo Pharmaceuticals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/hela+flp+in+pp1%CE%B3+2xstrep+cell+line/pmc11199634-356-5-17
Average 90 stars, based on 1 article reviews
hela flp-in pp1γ−2xstrep cell line - by Bioz Stars, 2026-09
90/100 stars
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purified pp1γ  (GloboZymes)
90
GloboZymes purified pp1γ
Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, <t>PP1γ,</t> and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.
Purified Pp1γ, supplied by GloboZymes, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/purified+pp1%CE%B3/pmc03610959-36-1-8
Average 90 stars, based on 1 article reviews
purified pp1γ - by Bioz Stars, 2026-09
90/100 stars
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gfp-pp1γ  (KU Leuven)
90
KU Leuven gfp-pp1γ
Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, <t>PP1γ,</t> and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.
Gfp Pp1γ, supplied by KU Leuven, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+%CE%B3/gfp+pp1%CE%B3/pm20206270-162-3-11
Average 90 stars, based on 1 article reviews
gfp-pp1γ - by Bioz Stars, 2026-09
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pp1γ crispr/cas9 ko plasmid  (Santa Cruz Biotechnology)
N/A
CRISPR/Cas9 KO Plasmids consists of PP1γ-specific 20 nt guide RNA sequences derived from the GeCKO (v2) library. For CRISPR gene knockout, gRNA sequences direct the Cas9 protein to induce a site-specific double strand break (DSB)
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pp1γ shrna (m) lentiviral particles  (Santa Cruz Biotechnology)
N/A
Gene Silencers generally consist of pools of three to five target-specific 19-25 nucleotide sequences in length. For independent verification of PP1γ gene silencing results, individual duplex components or plasmids are also available upon request. Suitable
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Image Search Results


NIPP1 inhibited PP1γ activity toward H3‐pThr 11 dephosphorylation. A, Venn diagram showing the number of PIPs that overlap among nucleus (GO:0 005 634), transcription, DNA‐templated (GO:0 006 351) and regulators of catalytic activity. Regulators of catalytic activity contain positive regulation of catalytic activity (GO:0 043 085), negative regulation of catalytic activity (GO:0 043 086), negative regulation of phosphatase activity (GO:0 010 923) and positive regulation of phosphoprotein phosphatase activity (GO:0 032 516). B, HCT116 cells were treated with UV and harvested at the indicated times. The chromatin fraction, soluble fraction, and whole cell extract (WCE) were subjected to immunoblotting using the indicated antibodies. IKKα and H3 were used as markers of soluble protein or chromatin protein, respectively. Relative band intensity of NIPP1 (WCE) was normalized by H3, compared with 0 h. Relative band intensity of NIPP1 on chromatin and soluble fractions was normalized by NIPP1 (WCE). Signals were quantified using ImageJ software. C, HCT116 cells were transfected with empty vector or vectors expressing myc‐his‐PNUTS. After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. D, Domain structure of NIPP1‐RATA and NIPP1‐ΔC mutant are shown. NIPP1 contains PP1‐binding region harboring the RVTF sequence (top). HCT116 cells were transfected with empty vector or vectors expressing the indicated NIPP1 (bottom). After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. Signals were quantified using Image Lab. E, HCT116 cells were transfected with either control, PNUTS, or NIPP1 siRNAs. After 70 h, the cells were treated with (+) or without (−) UV. Chromatin fractions were prepared for immunoblotting after 2 h incubation. Signals were quantified using Image Lab. F, In vitro phosphatase assay was performed using purified PP1γ and NIPP1. PP1γ‐WT, or T311A preincubated with his‐NIPP1 were incubated with chromatin. The phosphorylation of H3‐Thr 11 was monitored by immunoblotting using H3‐pThr 11 antibodies. The relative band intensity of H3‐pThr 11 was normalized by H3, compared with the control, and quantified using ImageJ software. G, HCT116 cells were treated with 25 J/m 2 UV and harvested at the indicated times (left). The chromatin fraction was subjected to immunoblotting using the indicated antibodies. H3 was used as markers of chromatin protein. Relative band intensity of NIPP1 was normalized by H3, compared with 0 h. Signals were quantified using Image Lab. Real‐time PCR analysis of CDK1 and CCNB1 expression in HCT116 cells (right). Data are provided as the mean ± SEM of 3 independent experiments. The results were considered statistically significant at * P < .05 and ** P < .01 [Correction added on 14 May 2021, after first online publication: Figure 1D has been corrected.]

Journal: Cancer Science

Article Title: PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage

doi: 10.1111/cas.14924

Figure Lengend Snippet: NIPP1 inhibited PP1γ activity toward H3‐pThr 11 dephosphorylation. A, Venn diagram showing the number of PIPs that overlap among nucleus (GO:0 005 634), transcription, DNA‐templated (GO:0 006 351) and regulators of catalytic activity. Regulators of catalytic activity contain positive regulation of catalytic activity (GO:0 043 085), negative regulation of catalytic activity (GO:0 043 086), negative regulation of phosphatase activity (GO:0 010 923) and positive regulation of phosphoprotein phosphatase activity (GO:0 032 516). B, HCT116 cells were treated with UV and harvested at the indicated times. The chromatin fraction, soluble fraction, and whole cell extract (WCE) were subjected to immunoblotting using the indicated antibodies. IKKα and H3 were used as markers of soluble protein or chromatin protein, respectively. Relative band intensity of NIPP1 (WCE) was normalized by H3, compared with 0 h. Relative band intensity of NIPP1 on chromatin and soluble fractions was normalized by NIPP1 (WCE). Signals were quantified using ImageJ software. C, HCT116 cells were transfected with empty vector or vectors expressing myc‐his‐PNUTS. After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. D, Domain structure of NIPP1‐RATA and NIPP1‐ΔC mutant are shown. NIPP1 contains PP1‐binding region harboring the RVTF sequence (top). HCT116 cells were transfected with empty vector or vectors expressing the indicated NIPP1 (bottom). After 48 h, the cells were treated with (+) or without (−) UV. The cells were harvested 2 h after treatment and chromatin fractions were subjected to immunoblotting using the indicated antibodies. Signals were quantified using Image Lab. E, HCT116 cells were transfected with either control, PNUTS, or NIPP1 siRNAs. After 70 h, the cells were treated with (+) or without (−) UV. Chromatin fractions were prepared for immunoblotting after 2 h incubation. Signals were quantified using Image Lab. F, In vitro phosphatase assay was performed using purified PP1γ and NIPP1. PP1γ‐WT, or T311A preincubated with his‐NIPP1 were incubated with chromatin. The phosphorylation of H3‐Thr 11 was monitored by immunoblotting using H3‐pThr 11 antibodies. The relative band intensity of H3‐pThr 11 was normalized by H3, compared with the control, and quantified using ImageJ software. G, HCT116 cells were treated with 25 J/m 2 UV and harvested at the indicated times (left). The chromatin fraction was subjected to immunoblotting using the indicated antibodies. H3 was used as markers of chromatin protein. Relative band intensity of NIPP1 was normalized by H3, compared with 0 h. Signals were quantified using Image Lab. Real‐time PCR analysis of CDK1 and CCNB1 expression in HCT116 cells (right). Data are provided as the mean ± SEM of 3 independent experiments. The results were considered statistically significant at * P < .05 and ** P < .01 [Correction added on 14 May 2021, after first online publication: Figure 1D has been corrected.]

Article Snippet: The antibodies used for immunoblotting or ChIP were Chk1 (sc8408 and sc56291; Santa Cruz), FLAG (M158‐3L; MBL), myc (sc789 and sc40; Santa Cruz), H3 (ab1791; Abcam), I‐2 (AF4719; R&D SYSTEMS), IKKα (sc7182; Santa Cruz), NIPP1 (612368; BD Biosciences; HPA027452; Sigma), normal goat IgG (sc2028; Santa Cruz), normal mouse IgG (sc2025; Santa Cruz), normal rabbit IgG (cs2729; Cell Signaling), PKAα (sc28315; Santa Cruz), PKA‐pThr 197 (cs4781; Cell Signaling), pS/T‐PKA (cs9624; Cell Signaling), H2AX (ab11175; Abcam), γH2AX (05‐636; Sigma), H3 (17168‐1‐AP; Proteintech), H3‐pThr 11 (ab5168; Abcam), PNUTS (611060; BD Biosciences), PP1‐pThr 321 (2581; Cell Signaling; Thr 321 corresponds to Thr 311 of PP1γ), PP1γ (sc6108; Santa Cruz; 07‐1298; Millipore), and MYPT1 (ab59235; Abcam).

Techniques: Activity Assay, De-Phosphorylation Assay, Western Blot, Software, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay, Sequencing, Incubation, In Vitro, Phosphatase Assay, Purification, Real-time Polymerase Chain Reaction

Phosphorylation of NIPP1 is important for the release of PP1γ A, Chromatin fractions from UV‐irradiated HCT116 cells were solubilized as shown in Materials and Methods and subjected to IP‐western analysis using the indicated antibodies. IgG was used as a negative control. The relative band intensity of NIPP1 was normalized by PP1γ. The results are shown compared with 0 h. Signals were quantified using Image J software. B, Domain structure and phosphorylation sites mediated by PKA and CK2 of NIPP1 are shown. NIPP1 contains PP1‐binding region harboring RVTF sequence. C, HCT116 cells were treated with TBB (50 μmol/L) or H89 (40 μmol/L) and irradiated with UV light. After 4 h, the cells were collected and the chromatin fractions were solubilized and subjected to pulldown with FLAG M2 agarose. Immunoblotting was performed using the indicated antibodies. The relative band intensity of PP1γ was quantified using Image Lab and normalized by that of FLAG‐NIPP1, compared with DMSO (n = 4). The results were considered statistically significant at * P < .05 and ** P < .01. D, HCT116 cells were transiently transfected with NIPP1‐WT, NIPP1‐S178A, or NIPP1‐S199A, lysed, and subjected to pulldown with FLAG M2 agarose. Immunoblotting was performed using the indicated antibodies. The relative band intensity of PP1γ was quantified using Image Lab and normalized by that of FLAG‐NIPP1, compared with NIPP1‐WT (n = 4). The results were considered statistically significant at * P < .05 and ** P < .01. E, HCT116 cells were transiently transfected with NIPP1‐WT, NIPP1‐S178A, or NIPP1‐S199A, lysed, and subjected to immunoblotting using the indicated antibodies

Journal: Cancer Science

Article Title: PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage

doi: 10.1111/cas.14924

Figure Lengend Snippet: Phosphorylation of NIPP1 is important for the release of PP1γ A, Chromatin fractions from UV‐irradiated HCT116 cells were solubilized as shown in Materials and Methods and subjected to IP‐western analysis using the indicated antibodies. IgG was used as a negative control. The relative band intensity of NIPP1 was normalized by PP1γ. The results are shown compared with 0 h. Signals were quantified using Image J software. B, Domain structure and phosphorylation sites mediated by PKA and CK2 of NIPP1 are shown. NIPP1 contains PP1‐binding region harboring RVTF sequence. C, HCT116 cells were treated with TBB (50 μmol/L) or H89 (40 μmol/L) and irradiated with UV light. After 4 h, the cells were collected and the chromatin fractions were solubilized and subjected to pulldown with FLAG M2 agarose. Immunoblotting was performed using the indicated antibodies. The relative band intensity of PP1γ was quantified using Image Lab and normalized by that of FLAG‐NIPP1, compared with DMSO (n = 4). The results were considered statistically significant at * P < .05 and ** P < .01. D, HCT116 cells were transiently transfected with NIPP1‐WT, NIPP1‐S178A, or NIPP1‐S199A, lysed, and subjected to pulldown with FLAG M2 agarose. Immunoblotting was performed using the indicated antibodies. The relative band intensity of PP1γ was quantified using Image Lab and normalized by that of FLAG‐NIPP1, compared with NIPP1‐WT (n = 4). The results were considered statistically significant at * P < .05 and ** P < .01. E, HCT116 cells were transiently transfected with NIPP1‐WT, NIPP1‐S178A, or NIPP1‐S199A, lysed, and subjected to immunoblotting using the indicated antibodies

Article Snippet: The antibodies used for immunoblotting or ChIP were Chk1 (sc8408 and sc56291; Santa Cruz), FLAG (M158‐3L; MBL), myc (sc789 and sc40; Santa Cruz), H3 (ab1791; Abcam), I‐2 (AF4719; R&D SYSTEMS), IKKα (sc7182; Santa Cruz), NIPP1 (612368; BD Biosciences; HPA027452; Sigma), normal goat IgG (sc2028; Santa Cruz), normal mouse IgG (sc2025; Santa Cruz), normal rabbit IgG (cs2729; Cell Signaling), PKAα (sc28315; Santa Cruz), PKA‐pThr 197 (cs4781; Cell Signaling), pS/T‐PKA (cs9624; Cell Signaling), H2AX (ab11175; Abcam), γH2AX (05‐636; Sigma), H3 (17168‐1‐AP; Proteintech), H3‐pThr 11 (ab5168; Abcam), PNUTS (611060; BD Biosciences), PP1‐pThr 321 (2581; Cell Signaling; Thr 321 corresponds to Thr 311 of PP1γ), PP1γ (sc6108; Santa Cruz; 07‐1298; Millipore), and MYPT1 (ab59235; Abcam).

Techniques: Irradiation, Western Blot, Negative Control, Software, Binding Assay, Sequencing, Transfection

PKA regulates the interaction between NIPP1 and PP1γ. A, HCT116 cells were cultured in the presence of Dox for 3 d to knockdown PKAα or luciferase (shControl) using tetracycline‐inducible shRNA and irradiated with UV. After the indicated times, the cells were collected and the total cell extracts were subjected to immunoblotting using the indicated antibodies. Signals were quantified using Image Lab. B, HCT116 cells expressing shControl or shPKAα were cultured with or without TBB, and treated with or without UV for 4 h. NIPP1 was immunoprecipitated from the total cell extracts and immunoblotted with an anti‐phospho‐S/T‐PKA substrate and anti‐NIPP1 antibodies. IgG was used as a negative control. C, HCT116 cells were transiently transfected with NIPP1‐WT or NIPP1‐S199A and cultured for 2 d. After irradiation with UV for 4 h, the cells were lysed and subjected to pulldown with FLAG M2 agarose. Immunoblotting was performed using the indicated antibodies. D, HCT116 cells were transfected with FLAG‐NIPP1 and cultured for 2 d. The cells were treated with 8Br‐cAMP (500 μmol/L) for 2 h and irradiated with UV. After 4 h, the total cell extracts were subjected to pulldown with FLAG M2 agarose. Samples were immunoblotted with the indicated antibodies. The relative band intensity of PP1γ and pS/T‐PKA was quantified using Image Lab and normalized to that of FLAG compared with control. E, An enrichment plot of HALLMARK_E2F_TARGETS in mouse 3T3 cells treated with H89 for 3 h compared with untreated 3T3 cells was generated using GSEA software. NES: normalized enrichment score; FDR: false discovery rate. Two biological replicates were analyzed. F, An enrichment plot of E2F1_BINDING_SCORE_ OVER_750 in mouse 3T3 cells treated with H89 for 3 h compared with untreated 3T3 cells was generated by GSEA software. E2F1 target gene set containing E2F1 binding scores higher than 750 in E2F target genes (n = 49). <xref ref-type= 32 NES: normalized enrichment score; FDR: false discovery rate. Two biological replicates were analyzed [Correction added on 14 May 2021, after first online publication: Figure 4C has been corrected.] " width="100%" height="100%">

Journal: Cancer Science

Article Title: PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage

doi: 10.1111/cas.14924

Figure Lengend Snippet: PKA regulates the interaction between NIPP1 and PP1γ. A, HCT116 cells were cultured in the presence of Dox for 3 d to knockdown PKAα or luciferase (shControl) using tetracycline‐inducible shRNA and irradiated with UV. After the indicated times, the cells were collected and the total cell extracts were subjected to immunoblotting using the indicated antibodies. Signals were quantified using Image Lab. B, HCT116 cells expressing shControl or shPKAα were cultured with or without TBB, and treated with or without UV for 4 h. NIPP1 was immunoprecipitated from the total cell extracts and immunoblotted with an anti‐phospho‐S/T‐PKA substrate and anti‐NIPP1 antibodies. IgG was used as a negative control. C, HCT116 cells were transiently transfected with NIPP1‐WT or NIPP1‐S199A and cultured for 2 d. After irradiation with UV for 4 h, the cells were lysed and subjected to pulldown with FLAG M2 agarose. Immunoblotting was performed using the indicated antibodies. D, HCT116 cells were transfected with FLAG‐NIPP1 and cultured for 2 d. The cells were treated with 8Br‐cAMP (500 μmol/L) for 2 h and irradiated with UV. After 4 h, the total cell extracts were subjected to pulldown with FLAG M2 agarose. Samples were immunoblotted with the indicated antibodies. The relative band intensity of PP1γ and pS/T‐PKA was quantified using Image Lab and normalized to that of FLAG compared with control. E, An enrichment plot of HALLMARK_E2F_TARGETS in mouse 3T3 cells treated with H89 for 3 h compared with untreated 3T3 cells was generated using GSEA software. NES: normalized enrichment score; FDR: false discovery rate. Two biological replicates were analyzed. F, An enrichment plot of E2F1_BINDING_SCORE_ OVER_750 in mouse 3T3 cells treated with H89 for 3 h compared with untreated 3T3 cells was generated by GSEA software. E2F1 target gene set containing E2F1 binding scores higher than 750 in E2F target genes (n = 49). 32 NES: normalized enrichment score; FDR: false discovery rate. Two biological replicates were analyzed [Correction added on 14 May 2021, after first online publication: Figure 4C has been corrected.]

Article Snippet: The antibodies used for immunoblotting or ChIP were Chk1 (sc8408 and sc56291; Santa Cruz), FLAG (M158‐3L; MBL), myc (sc789 and sc40; Santa Cruz), H3 (ab1791; Abcam), I‐2 (AF4719; R&D SYSTEMS), IKKα (sc7182; Santa Cruz), NIPP1 (612368; BD Biosciences; HPA027452; Sigma), normal goat IgG (sc2028; Santa Cruz), normal mouse IgG (sc2025; Santa Cruz), normal rabbit IgG (cs2729; Cell Signaling), PKAα (sc28315; Santa Cruz), PKA‐pThr 197 (cs4781; Cell Signaling), pS/T‐PKA (cs9624; Cell Signaling), H2AX (ab11175; Abcam), γH2AX (05‐636; Sigma), H3 (17168‐1‐AP; Proteintech), H3‐pThr 11 (ab5168; Abcam), PNUTS (611060; BD Biosciences), PP1‐pThr 321 (2581; Cell Signaling; Thr 321 corresponds to Thr 311 of PP1γ), PP1γ (sc6108; Santa Cruz; 07‐1298; Millipore), and MYPT1 (ab59235; Abcam).

Techniques: Cell Culture, Luciferase, shRNA, Irradiation, Western Blot, Expressing, Immunoprecipitation, Negative Control, Transfection, Generated, Software, Binding Assay

Schematic model of DNA damage‐induced transcriptional repression of E2F target genes. Under normal conditions, Chk1 phosphorylates H3‐pThr 11 , which induces K9 acetylation, leading to transcriptional activation of E2F1 target genes. PP1γ is inactivated through both Cdk‐dependent phosphorylation at pThr 311 and binding to NIPP1. In response to DNA damage, the ATR‐dependent release of Chk1 from chromatin indirectly suppresses Cdk activity, which results in the activation of PP1γ via a reduction in Thr 311 phosphorylation, and its release from NIPP1 is mediated by PKA phosphorylation. Activated PP1γ dephosphorylates H3‐pThr 11 in collaboration with Chk1 released from E2F1, ultimately resulting in the transcriptional repression of genes, such as CDK1 and CCNB1 , involved in the cell cycle

Journal: Cancer Science

Article Title: PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage

doi: 10.1111/cas.14924

Figure Lengend Snippet: Schematic model of DNA damage‐induced transcriptional repression of E2F target genes. Under normal conditions, Chk1 phosphorylates H3‐pThr 11 , which induces K9 acetylation, leading to transcriptional activation of E2F1 target genes. PP1γ is inactivated through both Cdk‐dependent phosphorylation at pThr 311 and binding to NIPP1. In response to DNA damage, the ATR‐dependent release of Chk1 from chromatin indirectly suppresses Cdk activity, which results in the activation of PP1γ via a reduction in Thr 311 phosphorylation, and its release from NIPP1 is mediated by PKA phosphorylation. Activated PP1γ dephosphorylates H3‐pThr 11 in collaboration with Chk1 released from E2F1, ultimately resulting in the transcriptional repression of genes, such as CDK1 and CCNB1 , involved in the cell cycle

Article Snippet: The antibodies used for immunoblotting or ChIP were Chk1 (sc8408 and sc56291; Santa Cruz), FLAG (M158‐3L; MBL), myc (sc789 and sc40; Santa Cruz), H3 (ab1791; Abcam), I‐2 (AF4719; R&D SYSTEMS), IKKα (sc7182; Santa Cruz), NIPP1 (612368; BD Biosciences; HPA027452; Sigma), normal goat IgG (sc2028; Santa Cruz), normal mouse IgG (sc2025; Santa Cruz), normal rabbit IgG (cs2729; Cell Signaling), PKAα (sc28315; Santa Cruz), PKA‐pThr 197 (cs4781; Cell Signaling), pS/T‐PKA (cs9624; Cell Signaling), H2AX (ab11175; Abcam), γH2AX (05‐636; Sigma), H3 (17168‐1‐AP; Proteintech), H3‐pThr 11 (ab5168; Abcam), PNUTS (611060; BD Biosciences), PP1‐pThr 321 (2581; Cell Signaling; Thr 321 corresponds to Thr 311 of PP1γ), PP1γ (sc6108; Santa Cruz; 07‐1298; Millipore), and MYPT1 (ab59235; Abcam).

Techniques: Activation Assay, Binding Assay, Activity Assay

Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, PP1γ, and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Differential localizations of protein phosphatase 1 isoforms determine their physiological function in the heart

doi: 10.1093/abbs/gmy171

Figure Lengend Snippet: Deletion of PP1β from mouse heart leads to increased HDAC7 phosphorylation (A–C) Western blot analysis for phospho-HDAC7, HDAC7, PP1α, PP1β, PP1γ, and GAPDH from the hearts of the indicated mice at 2 months of age. Phosphorylation of MLC2 was detected using a ProQ-Diamond staining method . (D) Quantification of western blots shown in A–C. N = 4 for each group. * P < 0.05 vs. NKX-Cre.

Article Snippet: For adenovirus infection, neonatal myocytes were first switched to serum-free medium and then incubated with purified adenoviruses expressing either β-galactosidase [ ] or PP1α (SL112646; SignaGen Laboratories, Rockville, USA), PP1β (SL112778; SignaGen Laboratories), PP1γ (SL112648; SignaGen Laboratories) for 2 h to enhance infection efficiency.

Techniques: Western Blot, Staining