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Fig. 3. The specific PXXP core motif within the PRR of BPGAP1 is indispensable for targeting the SH3 domain of EEN. (A) The three deletion mutants and two site-directed point mutants of BPGAP1. P1, devoid of the entire PRR; P2, retaining sequence PPPT; P3, retaining sequence KTPPPRPPLP; PP, substitution of the proline residues at 184 and 186 with alanines (underlined); P422A, substitution of proline residue at 422 with alanine (underlined). GST-recombinants of these deletion constructs (B) and site-directed mutants (C) were prepared as sepharose beads and used for pull-down assays using 293T cell lysates expressing endogenous EEN. Beads from the pull-down experiments were washed and processed for western analysis using EEN antibody. The antibody specifically detected endogenous EEN at 46 kDa, the expected mobility as verified using the overexpressed Flag-tagged EEN in the cells. (D) Cells were transfected with either the wildtype FL or PP mutant of Flag-tagged BPGAP1 and immunoprecipitated with anti-Flag M2 beads, washed and analyzed for bound endogenous EEN using EEN antibody. (E) Purified EEN from thrombin-cleaved GST recombinant was incubated with sepharose beads conjugated with GST fusions of full-length wildtype, the PP mutant of BPGAP1, or GST control, and bound targets revealed by western blot analyses using EEN antibody. WCL, whole cell lysates. FL, full-length.

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