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. 2003 Sep;23(18):6520–6532. doi: 10.1128/MCB.23.18.6520-6532.2003

FIG. 3.

FIG. 3.

Involvement of the PAI-2 C-D interhelical region and the C-pocket of Rb in Rb-PAI-2 binding. (A) Summary of data presented in panels B to E. +++, strong binding; +, weak binding; NT, not tested. (B) PAI-2 binds Rb and p130 but not p107. An in vitro GST binding assay used IVT, 35S-radiolabeled PAI-2 and the GST fusion proteins Rb379-928, p107385-1068, and p130414-1135. Input IVT PAI-2 (lane 3) was incubated with glutathione-agarose beads bound to either GST alone (lane 1), GST-Rb379-928 (Rb), GST-p107385-1068 (p107), or GST-p130414-1135 (p130). After washing, bead-bound, 35S labeled PAI-2 was resolved by SDS-PAGE and exposed to X-ray film (lane 2). (C) Binding of PAI-2 and the C-D interhelical deletion mutant of PAI-2 to Rb and Rb LXCXE mutants. Input IVT PAI-2 (lane 5), in the form of either 35S-labeled IVT PAI-2 (top panel) or a 35S-labeled IVT C-D interhelical deletion mutant of PAI-2 (C-D PAI-2) (bottom panel), was incubated with glutathione-agarose beads bound to either GST-Rb379-928 (lane 1), GST-Rb(706F) (Rb379-928 with a mutation in position 706, which disrupts LXCXE binding) (lane 2), or GST-Rb(del exon 21) (Rb379-928 with a deletion in exon 21, which also disrupts the LXCXE binding site) (lane 3), or GST alone (GST) (lane 4). After washing, bead-bound, 35S-labeled PAI-2 or C-D PAI-2 was resolved by SDS PAGE and exposed to X-ray film. (D) The RSL mutant of PAI-2 retains binding to Rb. Input IVT PAI-2 (lane 3, top panel), a C-D interhelical deletion mutant of PAI-2 (C-D PAI-2) (middle panel), or the RSL mutant of PAI-2 (PAI-2Ala380) was incubated with GST-Rb379-928. After washing, the bead-bound, 35S-labeled PAI-2 proteins were resolved by SDS-PAGE and exposed to X-ray film (lane 2). (E) PAI-2 binds to the C-pocket of Rb. Input 35S-labeled IVT PAI-2 (top panel, lane 6) or 35S-labeled C-D PAI-2 (bottom panel, lane 6) was incubated with glutathione-agarose beads bound to either GST (lane 1), GST-Rb379-928 (lane 2), GST-SE (lane 3), GST-SEΔ (lane 4), or GST-13S (lane 5). SE and GST-SEΔ bind c-Abl, but 13S does not. After washing, bead-bound, 35S-labeled PAI-2 or C-D PAI-2 deletion mutant PAI-2 was resolved by SDS-PAGE and exposed to X-ray film.