FIG. 7.
In vitro dephosphorylation of wild-type Rex-2, the S151D,S153D mutant, and mutant M17. (A) 293 T cells were transfected with 25 μg of wild-type (Wt) or rex mutant (S151D,S153D) expression vector. At 24 h posttransfection, cells were metabolically labeled using [35S]methionine-[35S]cysteine, and cell lysates were made as described in Materials and Methods. Transfected cell lysates were immunoprecipitated using anti-Rex-antiserum. Immune complexes were collected using protein A-Sepharose and incubated with 600 U of BAP for 3 h or mock treated, and the proteins were resolved by SDS-PAGE and visualized by autoradiography. 14C low-molecular-mass markers are indicated on the left (lane M). The results indicate that the phosphorylation-induced change in mobility on the SDS gel is due to the presence of aspartic acid residues at positions 151 and 153. (B) An experiment was performed as described for panel A to compare Wt Rex to mutant M17. The results indicate that the p26rex conformation of mutant M17 is the result of phosphorylation.