prosci anti puma  (ProSci Incorporated)

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 1: p38 activation, Tip60-T158 phosphorylation, and p53-K120 acetylation are induced with same kinetics by DNA damage. (A) Weston blot analysis of U2OS cells treated with 1 μM of Dox for indicated durations, detecting p38α, p–p38, p-Tip60-T158, Tip60, ac-p53-K120, p53 and actin. (B) Weston blot analysis of U2OS cells treated with indicated concentrations of Dox for 24 h, detecting p38α, p-p38, p-Tip60-T158, Tip60, ac-p53-K120, p53 and actin. (C) Weston blot analysis of U2OS cells treated with 1 μM Dox for indicated durations, detecting PUMA, p21 and actin. (D) Weston blot analysis of U2OS cells treated with indicated concentrations of Dox for 24 h, detecting PUMA, p21 and actin.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Activation Assay, Phospho-proteomics

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 2: p38α is required for the induction of Tip60-T158 phosphorylation, p53-K120 acetylation and p21WAF1 and PUMA expression following DNA damage. (A) U2OS cells transduced with shRNA for GFP (shGFP) or p38α (shp38α-756 or -758) were treated with 1 μM of Dox for 36 h (left panels) or 10 Gy of γ-radiation followed by incubation for 72 h (right panels). Cell lysis was subjected to Western blot analysis detecting the indicated proteins. (B) U2OS cells were pre-treated with 10 μM of the p38 inhibitor SB203580 or DMSO for 1 h, and then treated in the presence of SB203580 or DMSO with 1 μM of Dox for 36 h (left panels) or 10 Gy of γ-radiation followed by incubation for 72 h (right panels). Cell lysis was subjected to Western blot analysis detecting the indicated proteins. (C–D) U2OS cells transduced with shRNA for GFP (shGFP) or p38α (shp38α-756 or -758) were treated with 1 μM of Dox for 36 h (C) or 10 Gy of γ-radiation followed by incubation for 72 h (D) mRNA levels of PUMA (lower panels) and p21WAF1 (upper panels) were detected by real-time PCR. Values are mean ± SEM for triplicates. (E) U2OS cells were pre-treated with 10 μM of the p38 inhibitor SB203580 or DMSO for 1 h, and then treated in the presence of SB203580 or DMSO with 10 Gy of γ-radiation followed by incubation for 72 h. mRNA levels of PUMA (lower panel) and p21WAF1 (upper panel) were detected by real-time PCR. Values are mean ± SEM for triplicates.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Phospho-proteomics, Expressing, Transduction, shRNA, Incubation, Lysis, Western Blot, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 3: Tip60 is required for acetylation of p53 at K120 and induction of PUMA expression, but not for induction of p21WAF1, in response to DNA damage. (A) U2OS cells transduced with shRNA for GFP (shGFP) or Tip60 (shTip60-887 or -1506) were treated with 1 μM of Dox for 36 h (left panels) or 10 Gy of γ-radiation followed by incubation for 24 h (right panels). Cell lysis was subjected to Western blot analysis detecting the indicated proteins. (B–C) U2OS cells transduced with shRNA for GFP (shGFP) or Tip60 (shTip60-887 or -1506) were treated with 1 μM of Dox for 36 h (B) or 10 Gy of γ-radiation followed by incubation for 24 h (C). mRNA levels of PUMA (lower panels) and p21WAF1 (upper panels) were detected by real-time PCR. Values are mean ± SEM for triplicates. (D) U2OS cells transduced with shRNA for GFP (shGFP) or p53 (shp53) were left untreated (–) or treated with 1 μM of Dox for 24 h (+), and analyzed by Western blotting detecting the indicated proteins.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Expressing, Transduction, shRNA, Incubation, Lysis, Western Blot, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 6: p38α and Tip60 are essential for DNA damage-induced binding of p53 to the PUMA promoter. (A–B) U2OS cells transduced with shRNA for GFP (shGFP) or p38α (shp38α-756 or -758) were treated with 10 Gy of γ-radiation (10Gy) or left untreated (Ctrl) followed by incubation for 72 h. Cells were lysed and subjected to ChIP using normal mouse IgG (IgG) or a mouse anti-p53 antibody (p53). Immunoprecipitated DNA was used as the template for real-time PCR quantification of the p53-binding sites on the p21WAF1 (A) or PUMA (B) promoters. Values are mean ± SEM for triplicates. (C–D) U2OS cells transduced with shRNA for GFP (shGFP) or Tip60 (shTip60-887 or -1506) were treated 10 Gy of γ-radiation (10Gy) or left untreated (Ctrl) followed by incubation for 24 h. Cells were lysed and subjected to ChIP using normal mouse IgG (IgG) or a mouse anti-p53 antibody (p53). Immunoprecipitated DNA was used as the template for real-time PCR quantification of the p53-binding sites on the p21WAF1 (C) or PUMA (D) promoters. Values are mean ± SEM for triplicates.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Binding Assay, Transduction, shRNA, Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 7: Activated p38α directly phosphorylates Tip60-T158 in vitro and induces Tip60-T158 phosphorylation, p53-K120 acetylation, PUMA expression and apoptosis in cells. (A) Immunoprecipitation-coupled Kinase Assays for p38α. HA-p38α was immunoprecipitated from U2OS cells transduced with HA-p38α and treated with 1 μM of Dox for 36 h (left panels) or 10 Gy of γ-radiation followed by incubation for 48 h (right panels), and then incubated with recombinant Tip60α in the presence of cold ATP. Immunoprecipitated HA-p38α and Tip60-T158 phosphorylation were detected by Western blot using an anti-HA antibody and an anti-Tip60pT158 antibody, respectively. Input of recombinant Tip60α was stained by Ponceau S. (B) Western blot analysis of U2OS cells transduced with MKK3E, MKK6E or vector (Babe-puro), detecting MKK3, MKK6, p38α, p38β, p-p38, p-Tip60-T158, Tip60, ac- p53-K120, p53, PUMA, p21WAF1 and actin. Cells were lysed on day 3 post MKK3/6E transduction after selection of transduced cells. (C) Western blot analysis of U2OS cells transduced with wild-type (p38αWT, p38βWT) or indicated active mutant of p38 isoforms (p38αD179A, p38βD179A) or vector (Babe-puro), detecting p38α, p38β, p-p38, p-Tip60-T158, Tip60, ac-p53-K120, p53, PUMA, p21 and actin. Cells were lysed on day 3 post p38 transduction after selection of transduced cells. (D) FACS analysis of U2OS cells transduced with MKK3E, MKK6E or vector. Cells were collected on day 3 post MKK3/6E transduction after selection of transduced cells, and stained with a FITC-conjugated anti-Annexin-V antibody and FVD eFlour 660. (E) FACS analysis of U2OS cells transduced with wild-type (p38αWT, p38βWT) or indicated active mutant of p38 isoforms (p38αD179A, p38βD179A) or vector. Cells were collected on day 3 post p38 transduction after selection of transduced cells, and stained with a FITC-conjugated anti-Annexin-V antibody and FVD eFlour 660. (F) Quantification and statistical analysis of the data in D. The percentage of apoptotic cells was quantified as the percentage of FITC- positive cells in the gated area. Values are mean ± SEM for triplicates. (G) Quantification and statistical analysis of the data in E. The percentage of apoptotic cells was quantified as the percentage of FITC-positive cells in the gated area. Values are mean ± SEM for triplicates.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: In Vitro, Phospho-proteomics, Expressing, Immunoprecipitation, Transduction, Incubation, Recombinant, Western Blot, Staining, Plasmid Preparation, Selection, Mutagenesis

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 8: p38β is also essential for Tip60-T158 phosphorylation, p53-K120 acetylation, PUMA expression and apoptosis in response to DNA damage. (A) U2OS cells transduced with shRNA for GFP (shGFP) or p38β (shp38β-652 or -751) were treated with 1 μM of Dox for 36 h. Cell lysis was subjected to Western blot analysis detecting the indicated proteins. (B) U2OS cells transduced with shRNA for GFP (shGFP) or p38β (shp38β-652 or -751) were treated with 1 μM for 36 h. Cells were collected, stained with an FITC-conjugated anti-Annexin-V antibody and FVD eFlour 660 and analyzed by FACS. (C) Quantification and statistical analysis of the data in B. The percentage of apoptotic cells was quantified as the percentage of FITC-positive cells in the gated area. Values are mean ± SEM for triplicates.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Phospho-proteomics, Expressing, Transduction, shRNA, Lysis, Western Blot, Staining

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 9: p38α is essential for DNA damage-induced Tip60-T158 phosphorylation, p53-K120 acetylation, PUMA expression and apoptosis in primary human fibroblasts. (A) BJ cells transduced with shRNA for GFP (shGFP) or p38α (shp38α-756 or -758) were treated with 1 μM of Dox for 24 h. Cell lysis was subjected to Western blot analysis detecting the indicated proteins. (B) BJ cells transduced with shRNA for GFP (shGFP) or p38α (shp38α-756 or -758) were treated with 1 μM for 24 h. Cells were collected, stained with an FITC-conjugated anti-Annexin-V antibody and FVD eFlour 660 and analyzed by FACS. (C) Quantification and statistical analysis of the data in B. The percentage of apoptotic cells was quantified as the percentage of FITC-positive cells in the gated area. Values are mean ± SEM for triplicates.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Phospho-proteomics, Expressing, Transduction, shRNA, Lysis, Western Blot, Staining

Journal: Oncotarget

Article Title: Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage.

doi: 10.18632/oncotarget.2717

Figure Lengend Snippet: Figure 10: Phosphorylation of Tip60 at T158 by p38α is required for p53-K120 acetylation and PUMA induction after DNA damage. (A–B) U2OS cells were co-transduced with shRNA for GFP (shGFP) or Tip60 (shTip60-887 or -1506) and vector (WN), wild type mouse Tip60 (WT) or mutant mouse Tip60 (T158A). Cells were treated with 1 μM of Dox for 36 h (A) or 10 Gy of γ-radiation followed by incubation for 24 h (B). Cell lysis was subjected to Western blot analysis detecting the indicated proteins. (C–D) U2OS cells were co-transduced with shRNA for GFP (shGFP) or Tip60 (shTip60-887 or -1506) and vector (WN), wild type mouse Tip60 (WT) or mutant mouse Tip60 (T158A). Cells were treated with 1 μM of Dox for 36 h (C) or 10 Gy of γ-radiation followed by incubation for 24 h (D) mRNA levels of PUMA (right panels) and p21WAF1 (left panels) were detected by real-time PCR. Values are mean ± SEM for triplicates.

Article Snippet: The primary antibodies were from Cell Signaling (p38-pT180Y182, p38α and HA-tag), Santa Cruz (p53, p21WAF1), Prosci (PUMA), Abcam (acp53-K120) or Sigma (α-actin).

Techniques: Phospho-proteomics, Transduction, shRNA, Plasmid Preparation, Mutagenesis, Incubation, Lysis, Western Blot, Real-time Polymerase Chain Reaction