Rga3 is a Cdc42 GAP. (A) Medial-plane spinning-disk confocal images of Cdc42-mCherry^SW and CRIB-GFP in WT and rga3Δrga4Δrga6Δ. (B) The top graph shows average cortical profiles of Cdc42 and CRIB fluorescence intensity normalized to maximum and minimum values. For rga3Δrga4Δrga6Δ, only cells in which CRIB-GFP formed defined zones were used (n ≥ 20), so the described phenotype is underestimated in this quantification. For clarity, SDs are omitted from this graph. The bottom graph shows the nonnormalized CRIB-GFP fluorescence values for WT, rga3Δ, and triple rga3Δrga4Δrga6Δ mutants. Confidence bands show SD. n = 20 profiles per curve. (C) Medial-plane spinning-disk confocal images of Scd2-mCherry in WT and rga3Δrga4Δrga6Δ. Orange arrowheads indicate enlarged zones at cell poles; yellow arrowheads indicate zones extending over a large part of the cell cortex. (D) Cdc42-GTP pulldown assay. GST-CRIB was coupled to glutathione beads and mixed with extracts of WT, rga3Δ, rga4Δ, rga6Δ, or rga3Δrga4Δrga6Δ cells expressing Cdc42-mCherry^SW. Cdc42 was revealed in input and pulldown fractions with anti-mCherry antibody. One representative experiment is shown on top, and the average of three experiments is reported on the graph below. (E) In vitro GAP assay. [γ-³²P]GTP-loaded recombinant Cdc42 was incubated with recombinant full-length GAPs (WT or catalytically inactive; point mutations indicated) or with buffer, and Cdc42-associated radioactivity was determined by scintillation counting. Data are normalized to initial value. Bars, 3 µm. Error bars show SD.