Immunoprecipitation assays were performed as previously described [31 (link)]. Cells were washed twice with PBS, collected and homogenized with RIPA buffer. After cell debris was removed by centrifugation, extracts were aliquoted and either used immediately or stored at -80°C. Whole-cell lysates in lysis buffer were cleared with 1.0 μg nonimmune rabbit IgG (Santa Cruz) together with 30 μl of protein A-Sepharose beads (Pierce). After centrifugation, the lysates were immunoprecipitated for 1 h at 4°C with 1 μg of the anti-Stat6 antibody or nonimmune rabbit IgG and then incubated overnight at 4°C with protein A-Sepharose. The immunoprecipitates were washed three times with lysis buffer and once with PBS and then resuspended in electrophoresis sample buffer. Samples of immunoprecipitated or total proteins (30 μg) were analyzed by Western blotting using the anti-ppRB-Ser807/811 antibody (Cell Signaling Technology) against a pRB peptide phosphorylated on the Ser807/811 residue, which is phosphorylated by both CDK2 and CDK4/6 kinases [32 ], or the anti-pRB against underphosphorylated pRB (BD Biosciences-Pharmingen), the anti-PR antibody (abcam), anti-p21(abcam), anti-p27(abcam), and anti-GADPH (as control antibody). The blots represent typical results from at least three independent experiments.
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