Bca protein assay reagent

Whole cell lysates were prepared using RIPA lysis buffer containing complete protease inhibitor cocktail (Roche), homogenized and centrifuged at 12,000×g for 15 min at 4 °C. Protein concentration of cell lysates was determined by BCA protein assay reagent (TransGen). Cell lysates were incubated in SDS-PAGE sample loading buffer for 10 min at 98 °C, separated by 8%–12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), and incubated with primary antibodies specific for target proteins and horseradish peroxidase (HRP)-conjugated secondary antibodies. Super Signal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used for detection of protein of interest.

Total proteins were extracted from the
cells by radioimmunoprecipitation assay (RIPA) buffer containing protease
inhibitors (Macgene) and centrifuged at 12 000 rpm for 10 min
at 4 °C. The total protein concentration was determined by bicinchoninic
acid (BCA) protein assay reagent (TransGen). Protein samples were
transferred to a poly(vinylidene difluoride) (PVDF) membrane (Millipore)
after being separated by SDS-PAGE. Membranes were blocked with 5%
skim milk at 25 °C for 30 min and then incubated with primary
antibodies overnight at 4 °C. Subsequently, membranes were incubated
with horseradish peroxidase (HRP)-conjugated antirabbit or antimouse
IgG secondary antibody for 1 h at room temperature. The membranes
were analyzed by a Tanon-5200 Multi Gel Imaging Analysis System (Tanon)
and quantified by ImageJ software (ver. 1.8.0).