The cells and brain tissues were
collected and lysed with RIPA buffer (Solarbio, Beijing, China), with
added 1 mM PMSF and 1% cocktail of protease inhibitors (Solarbio,
Beijing, China). Protein concentrations were measured using the BCA
reagent (Beyotime, Shanghai, China). For Western blotting analysis,
an equal amount of protein was loaded for SDS-polyacrylamide gel electrophoresis.
Then, the proteins were transferred to polyvinylidene difluoride (PVDF)
membranes, which were then blocked with 5% skim milk for 2 h at room
temperature. Next, the membrane was incubated with the indicated primary
antibodies overnight at 4 °C. The membranes were then incubated
with HRP lgG (H + L) secondary antibodies for 2 h at room temperature.
Protein expression was visualized with an enhanced chemiluminescence
reagent (BeyoECL Star, Shanghai, China).
collected and lysed with RIPA buffer (Solarbio, Beijing, China), with
added 1 mM PMSF and 1% cocktail of protease inhibitors (Solarbio,
Beijing, China). Protein concentrations were measured using the BCA
reagent (Beyotime, Shanghai, China). For Western blotting analysis,
an equal amount of protein was loaded for SDS-polyacrylamide gel electrophoresis.
Then, the proteins were transferred to polyvinylidene difluoride (PVDF)
membranes, which were then blocked with 5% skim milk for 2 h at room
temperature. Next, the membrane was incubated with the indicated primary
antibodies overnight at 4 °C. The membranes were then incubated
with HRP lgG (H + L) secondary antibodies for 2 h at room temperature.
Protein expression was visualized with an enhanced chemiluminescence
reagent (BeyoECL Star, Shanghai, China).