HEK293T cells were transfected by the indicated plasmids. Thirty hours after transfection, protein was extracted using solution A [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, and 1× protease inhibitor (Roche)]. An aliquot of the extracts was immunoblotted with the indicated antibodies. The remaining extracts were immunoprecipitated using Sepharose beads bound to anti-His or anti-HA antibodies (Sigma-Aldrich) at 4°C overnight. After washing the Sepharose beads four times with solution B [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, and 1 mM PMSF], proteins were eluted by heating the beads to 98°C in 1× SDS–polyacrylamide gel electrophoresis loading buffer [50 mM tris-HCl (pH 6.8), 2% (v/v) SDS, 6% (v/v) glycerol, and 2% (v/v) β-mercaptoethanol]. The eluate was analyzed by immunoblotting with the indicated antibodies.

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