SH3BP1 associates with the epithelial junctional complex. (A–C) Caco-2 cells were processed for immunofluorescence with the indicated antibodies and then analyzed by epifluorescence (A and B) or confocal microscopy (C, xy and z sections are shown). For B, cells were transfected with siRNAs as indicated to determine the specificity of the SH3BP1 staining. (D) Frozen sections of mouse colon were stained with antibodies against SH3BP1 and ZO-1. Shown are confocal sections. The overlay also includes a nuclear stain in blue. (E) Junction formation by Caco-2 cells plated in low calcium was stimulated with calcium for the indicated periods to time. The cells were then fixed and stained for SH3BP1 and E-cadherin. Arrowheads mark early E-cadherin– and SH3BP1-positive junctions. (F) Caco-2 cells were stained for SH3BP1 after an incubation with the EGF receptor inhibitor PD153035 or, as a solvent control, DMSO. Bars, 10 µm.