Nrbp1

The cultured cells were treated with RIPA lysis buffer (50 mm Tris-HCl, pH 8.0, 1 mm ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate, 150 mm NaCl, 1% NP-40, 0.1% sodium deoxycholate) including cOmplete™ protease inhibitor mixture (Boster, Wuhan, Hubei, People’s Republic of China). The lysates were cleared by centrifugation (14,000 rpm) at 4°C for 20 minutes, and supernatants were collected as protein samples. Approximately 20–30 µg of each protein sample was run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). After blocking the nonspecific binding sites for 60 minutes with 5% nonfat milk, the membranes were incubated with primary antibodies against NRBP1 (GeneTex, at a 1:1,000 dilution), Cyclin D1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, at a 1:1,000 dilution), c-Myc (Santa Cruz Biotechnology Inc., at a 1:1,000 dilution), or β-actin (Boster, at a 1:10,000 dilution) overnight at 4°C. The membranes were then incubated with HRP-conjugated secondary antibody (at a 1:10,000 dilution) for 60 minutes at room temperature after three 10-minute washes with Tris-buffered saline and Tween 20 (TBST). The membranes were washed three more times with TBST and developed using an enhanced chemiluminescence system (Beyotime).