Western Blot Analysis of Cell Signaling

Western blotting was performed as previously described [38 (link),39 (link)]. All cell lysates (50–80 µg) were prepared in an ice-cold lysis buffer. Protein samples were loaded onto 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis membranes for electrophoretic separation and then transferred to PVDF membranes (Millipore) at 500 mA for 2 h. After blocking the buffer overnight with 5% non-fat dry milk in PBS containing Tween20 (PBST), the membranes were incubated with primary antibodies [Cyclin B1 (1:1000; Proteintech; 55004-1-AP), CDK1 (1:1000; Cell Signaling; E1Z6R), NRF2 (1:1000; Proteintech; 16396-1-AP), and β-actin (1:20,000; Sigma; A5441)] for 2 h at room temperature or overnight at 4 °C. Membranes were then washed once with PBST and twice with PBS, incubated with the secondary antibody Li-COR (Taipei, Taiwan) at a 1:20,000 dilution for 30–40 min, and washed again. Antigens were visualized using a near-infrared fluorescence imaging system (Odyssey LICOR, USA), and these data were interpreted using Odyssey2.1 software or a chemiluminescence detection kit (ECL; Amersham Corp., Arlington Heights, IL, USA). Densitometry analysis (including the integrated density of bands) was carried out using ImageJ (NIH), followed by normalization of the measured values to β-actin as a loading control.