FIGURE 3.

Specificity of Rac1b binding to p120^ctn. A–C, SCp2 mammary epithelial cells were transiently transfected with either YFP-Rac1b, YFP-Rac1, YFP-Rac1V12, or YFP as a negative control, and lysates were precipitated with GST-p120^ctn-4A (A) or the p120 deletion mutant lacking the polylysine sequence, GST-p120^ctn-4A-Δ (B) and Western blotted for YFP. C, Western blot (WB) of total cell lysates from SCp2 cells transiently transfected with YFP fusion proteins. D, GST-p120–4 was used to precipitate His-tagged Rac1, Rac1V12, Rac1N17, and Rac1b proteins that were nucleotide-free (−GDP/−GTPγS) or loaded with GDP or GTPγS, and precipitates were Western blotted and probed with Rac1 antibodies (top three blots) or GST antibodies (bottom two blots). E, immunofluorescence assay of mouse epithelial SCp2 cells transfected with YFP (left column), YFP-Rac1V12 (center column), or YFP-Rac1b (right column) stained with pan-p120 antibody, revealing that endogenous p120^ctn co-localizes with Rac1b both at cell-cell junctions and in the nucleus.