Western blot analysis
For Western blot analysis, cell samples were harvested and lysed by
adding 600 µl of RIPA Cell Lysis Buffer (P0013B, Beyotime) containing
0.6 mM PMSF. The lysate was centrifuged at 12,000 rpm for 15 min at 4°C
and the supernatant was collected. We added 5X SDS-PAGE protein loading
buffer to the collected protein samples at a ratio of 1:4. Heat in a
boiling water bath for 15 minutes to fully denature the protein. After
the sample was cooled to room temperature, we loaded the protein sample
directly into the sample well of the SDS-PAGE gel, and added 5-10ul to
each well, and kept constant voltage 80v electrophoresis for 1 hour.
Equal amounts of proteins were separated by SDS-PAGE and transferred to
PVDF membranes (Millipore). After blocking with 5% skim milk, the
membrane was incubated with the primary antibody overnight at 4°C,
followed by a horseradish peroxidase (HRP)-conjugated secondary antibody
for 2 hours at room temperature. The protein bands were visualized using
the ECL Ultra Sensitive Luminescence Kit (340958, Thermo), and the
analysis of the film bands was performed using Image J software.