FIG. 3.

PA157 protein is underphosphorylated. COS-1 cells were infected with vTF7-3 alone or in combination with VPA wt (vTF7-3+VPA) or VPA-T157A mutant (vTF7-3+VPA-T157A) and pulse labeled with ³²P_i for 2 h. Total cell extracts were analyzed by two-dimensional gel electrophoresis. The gels were transferred to nitrocellulose membranes and autoradiographed (top panels). The pH gradient from right to left is acidic to basic. The same membranes whose autoradiographs are shown were developed with MAbs specific for PA protein (bottom panels [Ab-PA]). The arrow shows the phosphorylation of the main PA isoform (marked by a solid lane) in wt PA, while the arrow indicates the absence of ³²P label in the most prominent PA isoform present in PA-T157A-infected cells.