Immunoprecipitation of Protein Complexes

Immunoprecipitation of proteins was done essentially as per published protocols25 (link)50 (link). Cells were lysed in a lysis buffer [20 mM Tris-HCl pH 7.4, 200 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 1 × EDTA-free protease inhibitor (Roche), 5 mM Vanadyl ribonucleoside comples (Sigma), 0.5% Triton X-100, 0.5% sodium deoxycholate] at 4 °C for 15 min, followed by clearing the lysate at 3,000 g for 10 min. Protein G agarose beads (Invitrogen) were blocked with 5% BSA in lysis buffer for 1 h and then incubated with required primary antibody for another 3–4 h before the lyaste was added. A final dilution of 1:50 (antibody:lysate) was used for immunoprecipitation. Immunoprecipitation was carried out for 16 h at 4 °C. Post washing with IP buffer (20 mM Tris-HCl pH 7.4, 150 mM KCl, 5 mM MgCl₂, 1 mM DTT, 1 × EDTA-free protease inhibitor (Roche)), the beads were divided in two halves: one subjected to RNA isolation with TRIzol LS and another for western blotting.