2.5 Western blot (WB) assay
293T cells (5×105 or 2×106) were
seeded in 6-well plates or 10 cm dishes 12 h before transfection.
Thirty-six hours post-transfection, the cells were lysed with
radioimmunoprecipitation assay (RIPA) buffer (Sigma) and cleared by
centrifugation. Then, the supernatants were collected, mixed gently with
loading dye, and boiled in a water bath for 5 min. Subsequently, the
samples were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to a polyvinylidene
difluoride (PVDF) membrane, which was blocked with 4% nonfat milk for 1
h at RT. After washing, the membrane was incubated with the primary
antibody and horseradish peroxidase (HRP)-conjugated secondary antibody.
Alternatively, the membrane was directly incubated with an
HRP-conjugated primary antibody. Finally, the PVDF membrane was exposed
to enhanced chemiluminescence (ECL).