Ab151579

Tissues were homogenized in an ice-cold RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride. The dissolved proteins were collected from the supernatant after centrifugation at 12,000 ×g for 20 min. Protein concentrations were determined using Coomassie blue-based assay reagent and then quantified. Protein extracts were separated by SDS-polyacrylamide gel electrophoresis and then electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% BSA and then incubated at 4 °C overnight with respective primary antibodies for anti-CK-B antibody (ab151579, Abcam, USA; 1:1000 in PBST), anti-CK-M antibody (ab151465, Abcam, USA; 1:1000 in PBST), anti-CK-S antibody (ab189314, Abcam, USA; 1:800 in PBST), and anti-β-actin (inner control, ab8227, Abcam, USA; 1:1500 in PBST). After washing with Tris-buffered saline Tween-20 (TBST), the membranes were incubated with a goat anti-rabbit secondary antibody conjugated to HRP for 1 h at room temperature. The antibody reactive bands were visualized using enhanced chemiluminescence detection reagents and a gel imaging system (Tanon Science & Technology Co., Ltd., China).