Western Blot Analysis Protocol

Western blot analysis was conducted as previously described [66 (link), 67 (link)]. Briefly, whole-cell lysates were extracted in urea lysis buffer (8.8urea, 5NaH₂PO₄, 1Tris, pH 8.0) and stored at −80°C for future use. A total of 80 μg of protein was mixed with 6x Lamelli buffer and boiled for 10 minutes. Protein extracts were run on a 4-15% pre-cast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) for 1 hour at a constant voltage of 150 V. Proteins were then transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA) for 1 hour at a constant current of 350 mA. The primary antibodies were as follows: monoclonal anti-BRG1 antibody (sc-17796, 1:200, Santa Cruz Biotechnology); monoclonal mouse anti-p21 (556430, 1:500, BD Pharmingen, San Jose, CA, USA); polyclonal rabbit anti-p16 (10883-1-AP, 1:500, Protein Tech, Chicago, IL, USA). Glyceraldehyde 3-phosphate dehydrogenase antibody (GeneTex Inc., Irvine, CA, USA) was used as the loading control. Anti-mouse and anti-rabbit secondary antibodies were purchased from GE Healthcare (Buckinghamshire, England, UK). Western blots were developed using an ECL Prime Western blot detection kit (GE Healthcare) and were analyzed with ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).