FIG. 1.

Hypophosphorylated pRB is the biologically active form of pRB. (A) G₀ quiescent human PBLs and PHA-stimulated (24-h) early-G₁ PBLs labeled in vivo with [³²P]orthophosphate and immunoprecipitated pRB were analyzed by phosphorimaging (top) and immunoblotting (bottom). Note the comigration of unphosphorylated and hypophosphorylated pRB on SDS-PAGE (bottom). (B) Anti-E2F4 immunoblot analysis from G₀ and G₁ PBLs detects equal levels of E2F4 in both cell types. (C) Anti-E2F4 immunoprecipitation followed by anti-pRB (left) or anti-p130 (right) immunoblot analysis from G₀ and G₁ PBLs. pRB failed to coimmunoprecipitate with E2F4 from G₀ PBLs, while p130 was readily detectable in complex with E2F4. IP, immunoprecipitates; α, antibody; WCE, whole-cell extracts.