Confluent or subconfluent cells were harvested and washed with ice-cold PBS (twice). The whole procedures were carried out at 4°C (on ice) unless specified otherwise. Cells were resuspended in freshly prepared lysis buffer [20 mM tris (pH 7.5), 1% Triton X-100, 150 mM NaCl, and protease inhibitors aprotinin (5 mg/liter) and 0.1 mM PMSF] and kept on ice for 30 min, followed by centrifugation at 14,000 rpm for 15 min at 4°C. Cells were directly lysed in SDS sample buffer [50 mM tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, and 10% glycerol] to obtain total cell lysates.

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