Western blot analysis
The treated cells were harvested and lysed in RIPA buffer. In some
experiments, NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo
Fisher, USA) were used to isolate the nuclear and cytosolic proteins
according to the manufacturer’s instructions. Equal amounts of proteins
from each group were loaded onto a 10% SDS-PAGE gel. After
electrophoresis, the proteins were transferred to membranes, and the
membranes were blocked with 5% non-flat milk and incubated with primary
antibodies overnight at 4ºC. Subsequently, the proteins were incubated
with the corresponding HPR-conjugated secondary antibodies for 30 min at
37ºC and visualized and imaged using a chemiluminescence (ECL) detection
system.