Fig. 4. EEN binds to BPGAP1 via its SH3 domain. (A) EEN domains used in binding studies. NT, N-terminal (aa 1-308) and CT, C-terminal (aa 309-368). NT contains a BIN/amphiphysin/Rvsp (BAR) domain and a coiled-coil region; CT contains an SH3 domain. (B) GST-recombinants of various constructs of EEN were prepared as sepharose beads and used for pull-down assays on lysates prepared from 293T cells that expressed Flag-tagged full-length BPGAP1. Beads from the pull-down experiments were washed and processed for western analyses using the Flag antibody. The blot was stripped and stained with amido black to reveal loading of GST-recombinants. (C) Purified EEN SH3 fusion of maltose binding protein, MBP-EEN SH3 or the MBP control were incubated with sepharose beads conjugated to purified GST recombinant proteins of the full-length wildtype, the PP mutant of BPGAP1, or GST control, and bound targets revealed by western blot analyses using MBP antibody. Purified MBP and MBP-EEN SH3 were analyzed with MBP antibody and revealed intact targets used in the direct binding assays. The lower apparent molecular mass for MBP-EEN SH3 compared with the MBP alone was due to the removal of internal lacZ coding sequence upon cloning of the target insert. The blot was stripped and stained by amido black to verify loading of equal amounts of GSTs.