Eif3b

The cells with 80% confluence in 15 mm cell culture dishes were collected and lysed within 1ml RIPA buffer containing 1 × protease inhibitor cocktail and 1 × phosphatase inhibitor cocktail for 20 min on ice. The supernatant with protein were obtained by centrifugation at 13,000g for 15 min at 4^oC. One-tenth volume of lysate was taken out as Input and the left part was mixed with 50 μl of Protein A/G magnetic beads (88803, Thermo Fisher Scientific) and gently rotated at 4^oC for 1 h to reduce non-specific binding and background. Then, 500–1,000 μg pre-cleared samples were incubated with antibodies against FLAG (F3165, Sigma-Aldrich), eIF3a (3411S, Cell Signaling Technology), eIF3b (sc-137214, Santa Cruz Biotechnology), and IgG (12–371, Millipore) for 1h under gentle agitation followed by incubation with 25μl of pre-washed Protein A/G magnetic beads under rotation at 4°C overnight. The proteins associated with Protein A/G magnetic beads were collected by placing the tubes into a magnetic stand, washed 3 times with IP washing buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EDTA, 150 mM NaCl, 1% Triton-X, 0.2 mM sodium orthovanadate) and then subjected to Western blotting. For the Co-IP assays with RNase digestion, the lysates were digested with RNase A/T1 mix (EN0551, Thermo Scientific) at RT for 30 min and the RNA was extracted to validate digestion efficacy before IP.