Cells were grown in 6-well tissue culture plates until confluency reached ~80%. Cells were then washed twice with ice-cold PBS and lysed on ice with RIPA buffer. The crude lysates were transferred to prechilled tubes and centrifuged at 13K RPM for 15 min at 4 °C. Cleared cell lysates were denatured with SDS sample buffer and resolved (~25 ug) on a 4–20 gradient SDS acrylamide gels (Bio-Rad), followed by transfer to polyvinylidene difluoride membranes (Bio-Rad). Membranes were incubated in 5% bovine serum albumin in PBST buffer for 1 h at room temperature, followed by incubation with the primary antibody (as specified) overnight at 4 °C. Membranes were then washed 3 times with PBST buffer and incubated in the appropriate secondary antibody at room temperature for 1 h, and the signals were developed using the Western Lights chemiluminescence detection kit (PerkinElmer Life Sciences).
For co-immunoprecipitation assays, 500 μg of cellular extracts were incubated with appropriate primary antibodies or normal rabbit/mouse immunoglobin G (IgG) on a rotator at 4 °C overnight, followed by addition of Protein G for 2 h at 4 °C. Beads were then washed four times with RIPA buffer and protease inhibitor mixture. The immune complexes were subjected to SDS-PAGE and immune-blotting as described above.
For co-immunoprecipitation assays, 500 μg of cellular extracts were incubated with appropriate primary antibodies or normal rabbit/mouse immunoglobin G (IgG) on a rotator at 4 °C overnight, followed by addition of Protein G for 2 h at 4 °C. Beads were then washed four times with RIPA buffer and protease inhibitor mixture. The immune complexes were subjected to SDS-PAGE and immune-blotting as described above.