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. 2012 Mar 15;23(6):1129–1139. doi: 10.1091/mbc.E11-12-1018

FIGURE 1:

FIGURE 1:

GARP regulates the secretion of TGFβ1 by forming a complex with proTGFβ1 on the cell surface. (A) LAP is coexpressed with GARP on the cell surface. 293T cells were transfected with mock, FLAG-tagged GARP, FLAG-tagged GARP + proTGFβ1, or proTGFβ1. Surface expression of FLAG-GARP and LAP1 was measured using FACS. Numbers in each histogram show the mean fluorescence intensity. GARP markedly increased LAP1 expression on the cell surface. (B) LAP is associated with GARP. 293T cells were transfected with the indicated plasmids. The clarified lysates were immunoprecipitated with anti-LAP1 antibody. The resulting samples were subjected to Western blot analysis using an anti-FLAG antibody. One-fifteenth of the cell lysates used in IP were loaded as input. (C) GARP disulfide links to proTGFβ1. 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with the anti-LAP1 antibody, analyzed by 7.5% nonreduced SDS–PAGE, and Western blotted with a different LAP1 antibody. A 250-kDa band representing the GARP–proTGFβ1 complex was detected in the lysate prepared from GARP- and proTGFβ1- cotransfected cells. (D) Association of GARP and proTGFβ1 prevents the direct secretion of proTGFβ1 into the supernatant. 293T cells were transfected with the indicated plasmids. The supernatants were immunoprecipitated with the anti-LAP1 antibody and analyzed by 10% reducing SDS–PAGE.