Fig. 2. Localization of Rho3. (A) Rho3 was detected in the insoluble fraction of the cell homogenate. Homogenates of wild-type cells grown in YEA medium at 30°C were spun at 10,000 g to obtain the supernatant (S) and pellet (P) fractions. Each fraction was run on an SDS-15% polyacrylamide gel and subjected to CBB staining or immunoblot analysis using anti-Rho3p antibodies. Molecular weight markers (M) were run on the left lane: trypsin inhibitor (21 k), carbonic anhydrase (31 k), actin (42 k), BSA (68 k), phosphorylase b (94 k), ß-galactosidase (116 k), and myosin heavy chain (210 k). The arrowhead on the right indicates a band of Rho3. (B) Immunofluorescent localization of Rho3. rho3 null cells containing pREP81Rho3 grown in EMM medium without thiamine at 30°C were fixed and stained with Bodipy-phallacidin (green in merged images), anti-Rho3p antibodies (red in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. No Rho3 signal was detected in the cells grown in the presence of thiamine (data not shown). Bar, 5 µm.