Immunoprecipitation, GST/His pulldown, and immunoblotting analyses

Immunoprecipitation was performed by preincubation of 0.5 to 1 mg of protein lysates with 1 μg of antibody for 1 hour at 4°C, followed by the addition of 20 μl of protein A/G Sepharose beads for 3 hours. For GST and His pulldown assays, GST- and His-tagged proteins plus their interacting proteins were incubated for 3 hours with glutathione Sepharose beads (GE Healthcare) and Ni Sepharose beads (GE Healthcare), respectively. The immunocomplexes or GST/His pulldown complexes were washed with lysis buffer (1.5 mM MgCl2, 0.2% NP-40, 125 mM NaCl, 5% glycerol, 25 mM NaF, 50 mM tris-HCl, and 1 mM Na3VO4) three times at 4°C, followed by boiling in 5× loading buffer at 95°C for 3 min. The immunoblotting analyses were performed as described previously (57).

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