Preparation of cell extracts

Forty-eight hours after transfection, cells in 35 mm dish were washed twice with PBS and lysed with 50 μL of EBC lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40) supplemented with an inhibitor mix (1 ng/ml of aprotinin, 100 mM β-glycerophosphate, 1 mM NaF, 1 mM Na3VO4, 10 μg/ml of leupeptin, 10 μg/ml of pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) or 50 μL of NativePAGE sample buffer (Thermo Fisher Scientific) supplemented with 10% n-dodecyl-β-D-maltoside and the inhibitor mix described above. The cell extracts were cleared by centrifugation at 15,000 rpm for 15 min at 4 ºC. The protein concentration of the precleared extracts was measured with Pierce BCA protein assay kit (Thermo Fisher Scientific).