For immunoprecipitation assays, cells were washed with cold PBS and lysed with cold lysis buffer (50mM Tris-Cl, pH 7.4, 150mm NaCl, 1mm EDTA, 1% NP-40, 0.25% sodium deoxycholate and protease inhibitor mixture) at 4°C for 30 min. Cellular extracts were incubated with appropriate primary antibodies on rotator at 4°C overnight, followed by the addition of protein A/G sepharose beads for 2 h at 4°C. Beads were then washed five times with lysis buffer. The immune complexes were subjected to SDS–PAGE followed by IB with secondary antibodies.