Substrate chemiluminescence kit

Cells were harvested and then lysed in lysis buffer (Beyotime Biotechnology). Equal amounts of protein in cell lysates were separated by electrophoresis in 8%–12% SDS polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, USA). The blotted membranes were incubated with various primary antibodies overnight, then incubated with secondary antibodies. The protein bands on membranes were visualized using the substrate chemiluminescence kit (Millipore) as previously reported
[23] (link).

Protein lysates were prepared as described (Lener et al., 2009 (link)). Appropriate amounts of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes as described (Greussing et al., 2013 (link)). Proteins of interest were detected by incubating the membranes with appropriate antibodies. Following primary antibodies were used: monoclonal mouse anti-p53 (#SC-126 Santa Cruz Biotechnology, USA), polyclonal rabbit anti-Lamin B1 (#ab16048 Abcam, United Kingdom), monoclonal rabbit anti-p21 waf1/cip1 (#2947S Cell signaling technology, USA), polyclonal rabbit anti-pp53 (Serin15) (#9284S Cell signaling technology, USA), and monoclonal mouse anti-GAPDH - glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#SC-25778 Santa Cruz Biotechnology, USA). Appropriate polyclonal HRP-conjugated secondary antibodies were used (Dako Cytomation, Denmark). Detection was performed in a ChemiDoc Imaging system (Bio-Rad Laboratories, USA) using a chemiluminescence substrate kit (Merck Millipore, Germany). As positive controls protein lysates obtained from HDFs at passage 35 and Cisplatin-treated HDFs (33 μM) were used. Results were normalized to the loading control. Analysis of densitometry was performed in ImageJ software and the values were normalized to the loading control (GAPDH).