PP2 binding stabilizes Lck and prevents its ubiquitination. (A) COS-7 cells were transfected with LckY505F (Y505F) or LckW97A (W97A). Cells either were treated with GA alone or were pretreated with PP2 for 1 h followed by incubation with both PP2 and GA for 0, 1, 2, or 3 h. Cells were lysed and immunoblotted for Lck. (B) 293T cells were transfected with LckY505F (Y505F) and were either left untreated (−), treated with GA for 1.5 h, or treated with PP2 for 15 h. Lck was immunoprecipitated by using MAb 3A5, and immunoprecipitates (IP) were immunoblotted for Hsp90 (top) or Lck (bottom). Untransfected (−) cells were not treated (−) and were used as a control. (C) COS-7 cells were transfected with LckY505F together with HA-ubiquitin and then were left untreated or were treated with PP2 for 15 h. Lck immunoprecipitates were immunoblotted with antiubiquitin (upper gel) and with anti-Lck antibodies (lower gel).