Anti ha beads

SILAC labeling reagents (89982 and 89990) were purchased from Thermo Scientific and cells were labeled according to the manufacturer’s instructions. Tandem affinity purification (TAP) or anti-Flag immune-precipitation (IP) was described previously^{17 (link)}. Briefly, cells were lysed in lysis buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 7.5, 0.5% NP40, 5 mM EDTA, Roche EDTA-free protease inhibitor cocktail) at 4°C for 30 minutes, followed by centrifugation at 13,000×g for 15 minutes at 4°C. The supernatants were incubated with anti-Flag beads (Sigma, A2220) at 4°C for 3 hours. Beads were washed three times with lysis buffer and then once with HA buffer (150 mM NaCl, 10% glycerol, 50 mM Tris pH 7.5, 0.05% NP40, 1 mM EDTA, Roche EDTA-free protease inhibitor cocktail) followed by elution with Flag peptide (0.5 mg ml⁻¹) in HA buffer. FLAG elution samples were incubated with anti-HA beads (Santa Cruz Biotechnology, sc-7392AC) at 4°C for 2 hours. Beads were washed three times with HA buffer followed by elution with HA peptide (0.4 mg ml⁻¹) in HA buffer.