Restore stripping solution

Total cell lysates and nuclear extracts were prepared using ice cold RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL) and Nuclear Extraction Kit (Cayman), respectively. The protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as a standard. Cell lysates were separated on 8–12% SDS-PAGE and transferred onto Hybond ECL nitrocellulose (GE Healthcare) at 20 volt overnight at 4°C. The membranes were blocked at 4°C in PBST blocking buffer (5% BSA in PBS with 0.05% Tween 20, pH 7.4) for 8 h. Blots were analyzed with each antibody (Table 1) at a dilution of 1:1000 overnight at 4°C. After three washes with PBST, the blots were incubated with suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:10,000–25,000 for 1 h. The blots were washed again and the proteins of interest were detected by Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare) according to the manufacturer’s instructions, and the chemiluminescence signal was then visualized with X-ray film. For reprobing, blots were treated with Restore stripping solution (Thermo Scientific). The intensities of these bands were analyzed with Phoretix Gel Analysis Software (Nonlinear Dynamics, Newcastle upon Tyne, UK).

RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used to prepare cell lysates. The protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as a standard reference.
Cell lysates were first separated on 8%–12% SDS-PAGE and then electroblotting onto polyvinylidene difluoride (PVDF) membranes (Hybond-P, GE Healthcare, Pittsburgh, PA, USA) using CAPS buffer system (10 mM CAPS pH 10.5, 10% (v:v) methanol) at 20 volts overnight at 4 °C. The membranes were blocked in freshly made blocking buffer (5% skim milk in PBS with 0.05% Tween 20, pH 7.4) for 6–8 h at room temperature. Blots were probed with specific primary antibody (Table 1) at a dilution of 1:1000–1:5000 overnight at 4 °C. Suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:10,000 was then added and incubated for 1 h before enhanced chemiluminescence detection (Amersham ECL Prime Western Blotting Detection Reagents, GE Healthcare). Some blots were stripped with Restore stripping solution (Thermo Fisher Scientific) before reprobed with another primary antibody. The intensity of the band was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).