Protein was extracted by the Hypotonic Lysis protocol immediately after cells were harvested post-transfection. Cell pellets were resuspended in 0.5 μl of a hypotonic lysis buffer (20 mM HEPES pH 7.9, 2 mM MgCl2, 0.2 mM EGTA, 10% glycerol, 0.1 mM PMSF, 1 mM DTT) per 100 × 20 mm tissue culture plate. Cells were kept on ice for 5 min and then underwent a freeze-thaw cycle three times to ensure proper detergent-independent cell lysis. NaCl was then added to the extracts at a concentration of 0.4 M and subsequently incubated on ice for 5 mins and spun down at 14,000 × g for 15 min at 4 °C. In all, 500 μl of Hypotonic Lysis buffer supplemented with 0.2% NP-40 was added to 500 μl of the supernatant extract.
