Enhanced bca protein assay reagent

After treatment, cells were collected and washed by PBS for twice. Cells were homogenized with ice-cold NP-40 buffer for 30 min to provide the whole cell proteins. Nuclear and cytoplasmic proteins were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) accordance to the manufacturer’s protocol. The concentrations of total proteins, nuclear proteins, and cytoplasmic proteins were determined using enhanced BCA protein assay reagent (TransGen Biotech, Beijing, China), respectively. Equal amounts of proteins extracts were separated on 8–12% SDS–PAGE gels and subsequently transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 5% skimmed milk solution and then probed with diluted primary antibodies (1:1000) overnight at 4°C. After incubating with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody, protein bands were developed with enhanced chemiluminescence (ECL) substrate and visualized by Tanon 5200 Imaging Analysis System (Tanon, Shanghai, China). Relative protein levels were performed by densitometry analysis using Image J software.

BV2 microglial cells were lysed in ice-cold RIPA buffer for half an hour to extract whole proteins. Nuclear and Cytoplasmic Protein Extraction Kit (NanJing KeyGen, Nanjing, Jiangsu, China) was used for nuclear and cytoplasmic proteins extraction. Enhanced BCA protein assay reagent (TransGen, Beijing, China) was used for determination of protein concentration. Ten microliters of protein of each group were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After the membranes were blocked in 5% skim milk solution, the blots were incubated with diluted primary antibodies overnight at 4 °C. Then, anti-rabbit-IgG-HRP-conjugated or anti-mouse-IgG-HRP-conjugated antibodies were used as secondary antibodies. ECL signals were recorded by Tanon 5200 Imaging Analysis System (Tanon, Shanghai, China). Image J software was used for densitometry analysis of relative protein levels.