CDC42u and CDC42b share the same panel of binding partners. (A) Transcribed exons and translated carboxy-terminal protein sequences of ubiquitous (CDC42u) and brain CDC42 (CDC42b). (B) Western blots showing immunoprecipitation of GFP-tagged proteins; GFP-CDC42b CA (b-CA) and GFP-CDC42u CA (u-CA) from transfected HEK cells. Coimmunoprecipitation of mycPar6 and PKCζ are shown in the top panels and their respective expression levels in total cell lysate in the lower input panel. (C) Quantification of coimmunoprecipitated mycPar6 and PKCζ normalized to their input values. The graph shows data points and means ± SEM of three independent experiments. (D–F) Correlation analysis plots showing the fold change (in Log₂ units) in the number of peptides of a given protein coprecipitated with constitutively active (CA) CDC42b (x-axis) versus with CDC42u (y-axis). The number of peptides is normalized by the respective number obtained in GFP control immunoprecipitation. Proteomic screening was performed by applying loose filtering parameters to segregate the interactors of the CA screen into GEFs, GAPs, and effectors. We identified 29 effectors out of 40 known epithelial effector proteins of CDC42. N-WASP and PAR6 proteins have been highlighted in red (E), seven known GEFs (F), and five known GAPs (G). (D–F) Peptides used to calculate significance are ≥6, P value ≤0.05, and number of replicates = 4. (G) Spinning disk images of HEK cells overexpressing the indicated pEGFP-CDC42 constructs 24 h after transfection. Note that, in these conditions, CDC42u and CDC42b display similar localization pattern. Source data are available for this figure: SourceData F2.