FIGURE 3:

C417A mutation abolishes the surface expression of GARP. (A) 293T cells were transfected with the indicated plasmids. Surface FLAG-GARP and LAP1 expression were measured by FACS. (B, C) The GARP C417A mutant associates with proTGFβ1. 293T cells were transiently transfected with the indicated plasmids. The clarified lysates were immunoprecipitated with the indicated antibodies, subjected to reducing SDS–10% PAGE (B) or nonreducing SDS–7.5% PAGE (C), and blotted with an anti-LAP1 antibody. (D) The GARP C417A mutant prevents secretion of proTGFβ1. 293T cells were transfected with the indicated plasmids. The supernatants were immunoprecipitated with an anti-LAP1 antibody, subjected to reducing SDS–10% PAGE, and blotted with a different anti-LAP1 antibody.