Fig. 3. Lack of rescue by TC10 of the growth defect of a temperature-sensitive cdc42 mutation in S. cerevisiae. A, a cdc42 temperature-sensitive yeast strain or the CDC42 parental strain was transformed with a plasmid that expresses the LEU2 gene and either TC10 or human Cdc42 driven by the constitutive yeast alcohol dehydrogenase (ADH1) promoter. Cells were selected for leucine prototrophy at 23°C (permissive temperature) for 2–4 days and then examined for rescue of the cdc42 temperature-sensitive growth defect at 37°C (restrictive temperature). B, TC10 or Cdc42 transformants were grown under selection for leucine prototrophy in liquid culture to A {approx} 1. Cells were then pelleted, washed with PBS plus protease inhibitors, and resuspended in 10 mM sodium azide, and extracts were prepared by rapid boiling and glass bead rupture of the cell wall. Equal amounts of extract were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for TC10 or Cdc42 using our anti-TC10 antibody or a commercially available anti-Cdc42 antibody (Santa Cruz Biotechnology), and protein expression was detected by chemiluminescence.