Anti p53 monoclonal antibody

The following antibodies were used: anti-Bnip3 polyclonal antibody (3769, Cell Signaling Technology), anti-Actin monoclonal antibody (A5441, Sigma Aldrich), anti-LC3B antibody (PM036, Medical and Biological Laboratories, Co.), anti-Sox2 monoclonal antibody (ab92494, Abcam, for western blotting), anti-Sox2 polyclonal antibody (AF2018, R&D, for IF), anti-Oct4 polyclonal antibody (ab19857, Abcam), anti-Nanog polyclonal antibody (ab106465, Abcam), anti-γH2AX polyclonal antibody (CST7918, Cell Signaling Technology), anti-53BP1 polyclonal antibody (NB100-304, Bio Techne), anti-p-ATM (Ser1981) monoclonal antibody (CST13050, Cell Signaling Technology), anti-ATM monoclonal antibody (CST2873, Cell Signaling Technology), anti-p-p53(Ser15) monoclonal antibody (CST9284, Cell Signaling Technology), anti-p53 monoclonal antibody (CST2527, Cell Signaling Technology), anti-Rad51 monoclonal antibody (H00005888, Abnova), anti-p-AMPK(Thr172) (CST2531, Cell Signaling Technology), anti-p-AMPK (CST2532, Cell Signaling Technology), anti-SSEA-1 monoclonal antibody (SC-21702AF647, Santa Cruz Biotechnology), Alexa Fluor 488 donkey anti-rabbit IgG (A21206, Invitrogen Thermo Fisher Scientific), Alexa Fluor 594 donkey anti-mouse IgG (A 21203, Invitrogen Thermo Fisher Scientific), Alexa Fluor 647 donkey anti-goat IgG (A32849, Invitrogen Thermo Fisher Scientific).

Cells were lysed in a lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 50 mM NaF and 1 mM Na₃VO₄) with a protease inhibitor cocktail. Cell lysates were mixed with 5 ul of an anti-COX-2 polyclonal antibody (#sc-1745, Santa Cruz Biotechnology) or an anti-p53 monoclonal antibody (#2524, Cell Signaling Biotechnology) at 4 °C overnight. Then, it was mixed with 20 ul of Protein A/G PLUS-agarose beads (#sc-2003, Santa Cruz Biotechnology) for 2 h at 4 °C. The beads were collected, and the bead-bound proteins were subjected to western blot analysis using the anti-p53 monoclonal antibody (1:500) and the anti-COX-2 polyclonal antibody (1:500), respectively.

Cells were seeded on 18 mm coverslips and cultured overnight. Cells were then fixed with 3.7% formamide and permeabilized with 0.2% Triton X-100. After blocking with 2% bovine serum albumin, cells were incubated with both anti-COX-2 polyclonal antibody (1:100; #sc-1745, Santa Cruz Biotechnology) and anti-p53 monoclonal antibody (1:100; #2524, Cell Signaling Biotechnology, 1:100) at 4 °C overnight. Cells were then incubated with both TRITC-conjugated anti-goat IgG (1:200; #A-21432, Thermo Fisher Scientific) and FITC-conjugated anti-mouse IgG (1:200; #F6257, Sigma-Aldrich) for 1 h at room temperature. Cell images were taken with a confocal microscope (Carl Zeiss).