Cultures were grown to mid-exponential phase and harvested by centrifugation at 3000 rpm for 5 min. The cells were washed with sterile H2O and resuspended in 1 mL of lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl, and 20% glycerol), with one protease inhibitor cocktail per 50 ml (complete, EDTA-free tablet, Roche Diagnostics, Indianapolis, IN, USA) and 1 mM PMSF (EMD Chemicals, Gibbstown, NJ, USA). Cells were lysed by bead beating twice for 4 min with 7 min on ice between cycles. Lysates were recovered by piercing a hole in the bottom of each tube, placing each tube in a 14 ml conical tube, and centrifuging at 1300×g for three 5-min cycles, recovering the supernatant after each stage. The combined lysate was cleared by centrifugation at 21,000×g for 10 min at 4 °C. The protein concentrations were determined using the Bradford assay68. Anti-HA immunoprecipitations were done using Pierce HA-Tag IP/Co-IP kit (Thermo-Fisher Scientific, PI23610), as per manufacturer’s instructions.