Fig 7.

Mapping the pRb binding surface on CR3 in vitro. (A) CR3 associates with the large pocket of pRb (pRbABC) in a dose-dependent manner. GST-pRbABC (0.3 μM) was incubated with increasing amounts of purified CR3 (residues 39 to 98), and the amount of associated CR3 was determined by silver staining. (B) Representative gel image of CR3-pRb binding as quantified for panel C. (C) Patch 1 residues most significantly contribute to pRb binding. Purified wild-type CR3 or the indicated mutant was assessed for the ability to bind pRb in a GST pulldown assay using 0.3 μM GST-pRbABC and 0.6 μM CR3. Data are presented relative to wild-type protein and are representative of a minimum of three independent assays. **, P ≤ 0.001; *, P = 0.032. (D) Based on the in vitro analysis, patch 1 and patch 2 residues significantly contributed to pRb binding. Colored in blue are patch 1 residues Y52, N53, V55, F57, C59, S63, T64, and T72, as well as R77, E80, and D81 within patch 2.