Immunoblot (IB) and immunoprecipitation (IP) assays

Cells were lysed with IP lysis buffer (Cat#87788, Thermo Fisher Scientific) supplemented with protease/phosphatase inhibitors (Cat#5872S, Cell Signaling Technology). Lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Then the membranes were incubated with various primary antibodies at 4 °C overnight, followed by incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (Cat#34096, Thermo Fisher Scientific). To perform immunoprecipitation, appropriate amounts of WCL were incubated with primary antibodies (2–3 μg) overnight at 4 °C. Protein A/G sepharose beads (Cat#78610, Thermo Fisher Scientific) were then added and the incubation was continued for 3–4 h before 4× wash with IP lysis buffer. For western blot analysis, equal amounts of WCL or immunoprecipitate were resolved by SDS-PAGE and immunoblotted with indicated antibodies.