Antihuman β actin rabbit monoclonal antibody

The total protein was extracted from the KCs using RIPA lysis buffer and 1:100 dilution of a protease inhibitor cocktail (Sigma, USA). A total of 30 μg of the protein lysate was resolved electrophoretically on SDS-PAGE and blotted onto PVDF membrane (Immobilon, USA). After being blocked for 1 h in blocking buffer (5% nonfat dried milk and 0.1% Tween-20 in Tris-buffered saline [TBS]) and separately incubated with an antihuman cyclin D1 rabbit monoclonal antibody (1:1000) (Cell Signaling, USA), an antihuman Bcl-xL rabbit monoclonal antibody (1:1000) (Cell Signaling, USA), and an antihuman β-Actin rabbit monoclonal antibody (1:1000) (Cell Signaling, USA) for 2 h at room temperature, the blots were washed three times with TBST (0.1% Tween in TBS) and incubated for 1.5 h with goat antirabbit IgG/HRP (1:3000) (ZSJB-BIO, China) at room temperature, followed by washing three times with TBST. The signals were visualized with the enhanced chemiluminescence method and detected by Tanon 4200 chemiluminescence imaging analysis system (Tanon, China). The band density was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized against the density of the respective housekeeping β-Actin.

Cultured cells were lysed in radioimmunoprecipitation assay buffer containing proteinase inhibitors (sc-24948, Santa Cruz Biotechnology), and loaded on 12.5% polyacrylamide gel. Proteins were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were immunoblotted with antihuman ZNF132 rabbit polyclonal antibody (PA5-41096, 1:500 dilution, Invirtogen, Carlsbad, CA) or antihuman β-actin rabbit monoclonal antibody (13E5, 1:1000 dilution, Cell Signaling Technology, Danvers, MA) and reacted with horseradish peroxidase-conjugated goat antibody (antirabbit IgG, 7074, Cell Signaling Technology), followed by incubation with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).