Figure 3.

T320 phosphorylation in cells stably transfected with wild-type PP-1Cα or a PP-1CαT320A mutant. T cell hybridoma KMls were infected with retrovirus stocks expressing either ID4-tagged wild-type PP-1Cα (T320) or ID4-tagged PP-1Cα in which T320 was mutated to alanine (T320A). Single clones that expressed similar levels of ID4-tagged PP-1Cα or PP-1Cα T320A were analyzed in this experiment. (Left) Control asynchronous cells. (Right) Untransfected (−), wild-type PP-1Cα-transfected (T320), and PP-1Cα T320A-transfected (T320A) cells were synchronized at the G₂/M phase using 400 ng/ml nocodazole treatment for 16 h. Whole cell lysates were prepared as described and aliquots (20 μg protein) were resolved on SDS/PAGE, followed by immunoblotting with either G-97 antibody (A) or anti-PP-1Cα antibody (B). The ID4-tagged PP1C (T-PP-1Cα) migrated with a slightly higher molecular weight than endogenous PP1Cα.