Cells were lysed in lysis buffer [0.5% Triton X-100, 20 mM Hepes (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerolphosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 1 mM Na3VO4, and 2 mM dithiothreitol (DTT)] containing protease inhibitors. Lysates were centrifuged and incubated with indicated antibodies at 4°C overnight. The next day, prewashed protein A/G beads (Pierce) were added and incubated at 4°C for 4 hours. The beads were washed with cold phosphate-buffered saline (PBS) four times and eluted with DTT-containing SDS sample buffer by boiling for 10 min before immunoblotting.

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