Role of the fMAPK scaffold Bem4p in stabilization of Cdc42p levels. (A) Cdc42p-interacting proteins. Colors indicate Cdc42p levels by immunoblot analysis in cells lacking the indicated protein. Red, lower levels; green, higher levels; grey, not tested. (B) Cdc42p levels in wild-type cells (WT, PC538), and the bem1Δ (PC6680), bem4Δ (PC3351), and ste11Δ (PC3862) mutants using antibodies to Cdc42p. See Fig. 1 B for details. CDC42 mRNA levels were detected by RT-PCR from two independent replicates, SD < 0.1 for all strain tested. (C) Wild-type cells (WT, PC538) and bem4Δ mutant (PC3551) expressing GFP-Cdc42p (PC6454) were grown for 4 h at 30°C and examined by fluorescence microscopy. Micrographs were taken at the same exposure time. Scale bar, 5 µm. (D) Relative fluorescence intensity of GFP-Cdc42p in wild-type cells and the bem4Δ mutant. Error bars indicate SD among three biological replicates (n = 3); 50 cells were examined in each replicate. Data were analyzed using unpaired t test (two-sided). P refers to P value. (E) Levels of ubiquitinated GFP-Cdc42p in wild-type cells and the bem4Δ mutant. Colored circle refers to cells expressing the indicated versions of Cdc42p and Bem4p. See Fig. 1 A for details. (F) Serial dilutions of wild-type cells (WT) and bem4Δ mutant (PC3551) expressing GFP-Cdc42p (PC6454) on SD-URA media. (G) Levels of GFP-Cdc42p^Q61L (Q61L, PC7458) in wild-type cells (WT) and the bem4Δ mutant. See Fig. 1 B for details. (H) Localization of Gic2p-PBD-tdTomato in wild-type cells (WT, PC538) and the bem4Δ mutant (PC3551). Micrographs were taken at the same exposure. Scale bar, 5 µm. Source data are available for this figure: SourceData F4.