SH3BP1 forms a complex with CD2AP and the junctional scaffold JACOP. (A and B) Extracts from Caco-2 (A) and A431 (B) cells were subjected to immunoprecipitation with the indicated antibodies. An IgG control from the same species as each of the specific primary antibodies is shown. The JACOP antibody recognized only a weak band in total cell extracts, and coprecipitation of this protein could not be detected. (C) Caco-2 cells were fixed and processed for immunofluorescence using antibodies against SH3BP1, CD2AP, and JACOP. Shown are confocal xy and z sections. (D) Calcium was added to Caco-2 cells plated in low calcium for the indicated periods to time. The cells were then fixed and stained as indicated. (E) EGF-stimulated A431 cells were fixed and processed as indicated. Shown are epifluorescence images of the dorsal aspect of the cells. Note that CD2AP and JACOP colocalize with SH3BP1 in dorsal ruffles. (F) Caco-2 cells were stained for CD2AP or JACOP after an incubation with the EGF receptor inhibitor PD153035 or, as a solvent control, DMSO. (G) GST fusion proteins conjugated to glutathione beads were incubated with A431 lysate (NTD, N-terminal domain; CTD, C-terminal domain). Pull-down experiments were analyzed by immunoblotting. (H) A431 cells transfected with siRNAs as indicated were lysed, and coprecipitation of CD2AP and SH3BP1 with JACOP was analyzed. Note that depletion of SH3BP1 inhibits coprecipitation of CD2AP. Bars, 10 µm.