Pmsf r0010

Total proteins were isolated from tissues and cells by RIPA lysis buffer with phenylmethylsulfonyl uoride (PMSF; R0010; Solarbio Science & Technology, Beijing, China), and the total protein concentration was determined by a BCA protein assay kit (20201ES76; Yeasen Company, Shanghai, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene uoride (PVDF) membranes (FFP 36; Beyotime, Shanghai, China). 5% bovine serum albumin (BSA) was used to block the membranes for 2 h at 37 ℃. DLC1 rabbit polyclonal antibody (ab126257, 1:1000, Abcam, Cambridge, UK) and GAPDH rabbit polyclonal antibody (ab22555, 1:2000, Abcam) were added onto the membranes for incubation overnight at 4 °C. PBST (phosphate buffered saline buffer + 0.1% Tween-20) was used to wash the membranes three times with 10 min each time. Horseradish peroxidase (HRP)-labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1:3000, Abcam) was added onto the membranes for 1 h of incubation at room temperature and then the membranes were washed with PBST three times. Images of the protein bands were observed and captured under an optical luminometer (GE, USA).