Puma antibody

Protein was extracted from cardiac tissues or cell samples using RIPA lysis buffer. The cell lysate was fractionated by 10% SDS‐PAGE and subsequently transferred to PVDF membranes (Millipore) and incubated with different primary antibodies overnight. After incubated with the primary antibodies, membranes were washed with TBS‐T and incubated with the corresponding secondary antibodies for 1h at room temperature. Finally, the blots were scanned by a Bio‐Rad ChemiDoc XRS+system (Bio‐Rad). The following antibodies were used: Runx1 antibody (Cell Signaling Technology; #4334; dilution 1:1000), p53 antibody (Abcam; #ab26; dilution 1:1000), p‐p53 antibody (Abcam; #ab223868; dilution 1:2000), Noxa antibody (Cell Signaling Technology; #14766; dilution 1:1000), Bax antibody (Cell Signaling Technology; #5023; dilution 1:1000), Puma antibody (Cell Signaling Technology; #24633; dilution 1:1000), Anp antibody (Abcam; #ab181242; dilution 1:10 000) and Gapdh antibody (Abcam; #ab8245; dilution 1:50 000).

Western blotting analysis was as described in our previous article.²² (link) Briefly, 72 hr after transfection, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA), and then western blotting was carried out with standard procedures. The primary antibodies included SETDB1 antibody (1:1,000; Cell Signaling Technology, Boston, MA, USA), p53 antibody (1:1,000; Cell Signaling Technology, Boston, MA, USA), p21 antibody (1:1,000; Cell Signaling Technology, Boston, MA, USA), PUMA antibody (1:1,000; Cell Signaling Technology, Boston, MA, USA), Gadd45 antibody (1:1,000; Cell Signaling Technology, Boston, MA, USA), γ-H2AX antibody (1:1,000; Cell Signaling Technology, Boston, MA, USA), and GAPDH antibody (1:2,000; Santa Cruz Biotechnology). The secondary antibodies included anti-mouse horseradish peroxidase (HRP)-conjugated antibody (1:5,000; Bio-Rad, Hercules, CA, USA) or anti-rabbit-HRP (1:5,000; Cell Signaling Technology, Boston, MA, USA).