FIGURE 2:
Cys-4 of TGFβ1 disulfide links to GARP. (A) ProTGFβ1 C4S mutant is coexpressed with GARP on the cell surface. 293T cells were transfected with the indicated plasmids, and the surface FLAG-GARP and LAP1 expressions were measured by FACS. (B) ProTGFβ1 C4S mutant associates with GARP. 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with anti-FLAG or anti-LAP1 antibody, subjected to reducing SDS–10% PAGE, and blotted with a different anti-LAP1 antibody. (C) GARP disulfide links to Cys-4 of 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) C4S mutation reduces the stability of the GARP–proTGFβ1 complex. 293T cells were transfected with the indicated plasmids. The supernatants were immunoprecipitated with anti-LAP1 antibody, subjected to reducing SDS–10% PAGE, and blotted with a different anti-LAP1 antibody.