FIGURE 4:

Cys-192 and Cys-331 of GARP disulfide link to proTGFβ1. (A) 293T cells were transfected with the indicated plasmids, and the surface FLAG-GARP and LAP1 expressions were measured by FACS. (B) The mutated GARPs associate with proTGFβ1. The cell lysates were immunoprecipitated with anti-FLAG antibody, subjected to reducing SDS–10% PAGE, and blotted with the indicated antibodies. (C) Cys-192 and Cys-331 of GARP disulfide link to proTGFβ1. 293T cells were transfected with the indicated plasmids. The clarified lysates were immunoprecipitated with anti-LAP1 antibody, subjected to reducing SDS–7.5% PAGE, and blotted with a different anti-LAP1 antibody. (D) C192A/C331A double mutation in GARP reduces the stability of the GARP–proTGFβ1 complex. 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.