FIGURE 3.

Immunodetection of Thr-955 phosphorylation and bound 14-3-3 proteins for the wild-type PMA2 and the T889D and E14D mutants. A and C, the plasma membrane fraction (5 μg of protein) isolated from a 36-h culture (YD, pH 6.5) of yeast strains expressing wild-type or mutant PMA2 (A, left panel) or the corresponding His₆-tagged PMA2 purified by Ni²⁺-NTA chromatography (A, right panel, and C) was analyzed by SDS-PAGE and Western blotting using antibodies against PMA2 (top), PMA2 phospho-Thr-955 (pPMA2; middle), or 14-3-3 proteins (bottom). Note that the double band identified by anti-14-3-3 protein antibodies corresponds to the two S. cerevisiae 14-3-3 proteins. B and D, the PMA2 phospho-Thr-955/PMA2 signal ratio (white bars) and the 14-3-3 protein/PMA2 signal ratio (black bars). The signals were quantified using the Image Station 4000R and Molecular Imaging software from Eastman Kodak Co. and were normalized to that for PMA2 (set at 100%). The data are the mean and S.E. for seven independent experiments in B and four independent experiments in D. The statistical significance of the difference between the indicated results is shown by the asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001).