Anti phospho p53 ser15 polyclonal antibody

All antibodies used in this study are anti-human anti-p53 polyclonal antibody, anti-phospho-p53-(Ser15) polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA); anti-α-tubulin monoclonal antibody (BD Phamingen, San Jose, CA, USA); anti-PIG3 (H300) polyclonal antibody, anti-p53 (DO-1) monoclonal antibody, anti-MDM2 (SMP14) monoclonal antibody, anti-myc polyclonal antibody, anti-HA polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). We followed manufacturer’s protocol for dilution of all primary antibodies.

Cells were lysed in a lysis buffer (150 mM NaCl, 50 mM Tris–HCl, 1% NP-40, 0.25% sodium deoxycholate, and 0.1% SDS) with 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin and 1 µg/ml pepstatin. Proteins were resolved on 10% or 12% SDS–polyacrylamide gels and transferred to PVDF membranes, which were probed with specific antibodies. The immunoreactive protein complexes were detected by enhanced chemiluminescence (Amersham Bioscience, UK) (Choi et al., 2009 (link)). The specific antibodies used in this study were as follows: an anti-DNAJB9 polyclonal antibody raised against a synthetic peptide (Kurisu et al., 2003 (link); Lee et al., 2015 (link)), an anti-Pan-Ras monoclonal antibody (OP40; Calbiochem, USA), an anti-p53 polyclonal antibody (sc-6243; Santa Cruz Biotechnology, USA), an anti-phospho-p53 (Ser15) polyclonal antibody (#9284; Cell Signaling Technology, USA), an anti-p21^WAF1/CIP1 polyclonal antibody (sc-397; Santa Cruz Biotechnology), and an anti-β-actin monoclonal antibody (sc-81178; Santa Cruz Biotechnology).