FIGURE 6.

Immunodetection of Thr-955 phosphorylation and bound 14-3-3 proteins for purified PMA2 mutants expressed in N. tabacum BY2 culture cells. A, 3-day-old cultures of N. tabacum BY2 cells expressing the indicated wild-type or mutant His-tagged PMA2 were harvested and homogenized in the presence of 1 μm fusicoccin, and a microsomal fraction was prepared, and then the proteins were solubilized, and the tagged PMA2 was purified by Ni²⁺-NTA chromatography and analyzed by SDS-PAGE and Western blotting using antibodies against PMA2 (top), PMA2 phospho-Thr-955 (pPMA2; middle), or 14-3-3 proteins (bottom). B, 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 Kodak and were each normalized to that for PMA2 (set at 100%). The data are the mean and S.E. for four independent experiments. The statistical significance of differences between results is indicated by the letters, results with different letters being significantly different by analysis of variance (p < 0.05).