Connecdenn binds to SH3 domains from endocytic proteins. A, Soluble lysates from Flag–connecdenn-transfected HEK-293 cells were incubated with GST or GST fused to the SH3 domain of either amphiphysin I or II, PACSIN 1 or 2, endophilin A1, and the five individual SH3 domains of intersectin, precoupled to glutathione-Sepharose beads. Proteins specifically bound to the beads were processed for Western blot with antibodies against the indicated proteins. B, Soluble brain lysates were incubated with anti-intersectin serum or preimmune serum from the same rabbit along with protein A-Sepharose beads. Proteins specifically bound to the beads were processed for Western blot with antibodies against the indicated proteins. C, Soluble brain lysates were incubated with anti-connecdenn serum (3775) or preimmune serum from the same rabbit along with protein A-Sepharose beads. Proteins specifically bound to the beads were processed for Western blot with antibodies against the indicated proteins. For B and C, Western blots with polyclonal antibodies were revealed with protein A-HRP. For all experiments, an aliquot of starting material (SM) equal to 1/10 of that added to the beads was run in parallel.