FIGURE 7:

The α_Vβ₆-mediated TGFβ activation from the GARP–proTGFβ complex requires the RGD motif in LAP and membrane association of GARP. (A, B) α_Vβ₆ does not interfere with the interaction between GARP and proTGFβ1. Mock or α_Vβ₆-expressing cells were transfected with the indicated plasmids. The clarified lysates were immunoprecipitated with the indicated antibodies, subjected to reducing SDS–10% PAGE (A) or nonreducing SDS–7.5% PAGE (B), and blotted with an anti-LAP1 antibody. (C) An RGD peptide interferes with α_Vβ₆-mediated TGFβ activation. Mock or α_Vβ₆-expressing cells were transfected with the indicated plasmids and cocultured with TMLC in the presence of 0.5 mM RGE (GRGESP; control peptide) or RGD peptide (GRGDSP). (D, E) sGARP interacts with proTGFβ1. The extracellular domain of GARP was fused to a His-SBP tag to generate the soluble GARP construct. 293T cells were transfected with the indicated plasmids. The clarified lysates (D) and supernatants (E) were immunoprecipitated with our in-house GARP antibody (GARP2) or anti-LAP1 antibody and blotted with a different anti-LAP1 antibody. (F, G) Membrane association is required for GARP to support α_Vβ₆- or α_Vβ₈-mediated TGFβ activation. Cells were transfected with the indicated plasmids and cocultured with TMLC to assess active TGFβ production.