FIG. 5.
Vac17 is a phosphoprotein and CLA4-overexpressing cells exhibit a decrease in Vac17 levels. (A) Vac17-ProA and actin protein levels were examined by Western blot analysis in untagged (W303-1A) and VAC17-ProA (YCH4774) cells. (B) Vac17-ProA was immunoprecipitated from protein extracts of 4X GAL-CLA4t swe1Δ VAC17-ProA (YCH4893) or untagged (UT) 4X GAL-CLA4t swe1 (Piatti2711) strains grown on YP plus 2% galactose medium for 6 h. Immunoprecipitated material was split and treated with buffer alone, λ-phosphatase, or λ-phosphatase plus phosphatase inhibitors. (C and D) Mid-logarithmic-phase VAC17-ProA (YCH4774) and GAL-CLA4 VAC17-ProA (YCH4882) cells were grown in YP plus 2% raffinose. At the zero time point 2% galactose was added to induce CLA4 overexpression, samples were taken every hour, and Western blot analysis was performed. (E) Mid-logarithmic-phase GAL-STE20 VAC17-ProA (YCH4843) cells were grown in SC-TRP plus 2% raffinose. At the zero time point 2% galactose was added to induce STE20 overexpression, samples were taken every hour, and Western blot analysis was performed.