Antibodies against human p53 phosphorylated at ser15

Western blotting was carried out as described previously.²², ²³, ⁴⁵ Briefly, cells or tissue homogenates were lysed in NP40 cell lysis buffer (Biosource, Camarillo, CA) to extract total cellular proteins following these treatments. Equal amounts of proteins (50 µg/lane) were resolved by using 4‐20% gradient SDS‐PAGE (Life Technologies). The nitrocellulose‐membrane blots transferred were blocked in 5% fat‐free milk in 0.05% Tween‐20, 20 mM phosphate‐buffered saline, pH 7.4 (PBST), and then incubated with each one of the primary antibodies (1:500 or 1:5000 dilution), at 4ºC overnight. These blots were incubated with corresponding horseradish peroxidase‐conjugated secondary antibodies (1:5000 dilutions) and detected using SuperSignal West Femto substrate (Thermo Fisher Scientific). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control for cellular protein. Antibodies against human p53 phosphorylated at Ser15 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies for Puma, p21Waf1/Cip1 (p21), Bax, p53, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX). Relative protein levels were calculated from optical density values for each protein band, normalized against those for GAPDH from three separate blots.