GARP increases cleavage of the pro-TGF-β1 precursor and secretion of latent TGF-β1 in T lymphocytes.

<p>Cell lysates were analyzed by WB after SDS-PAGE under reducing conditions with antibodies against GARP, β-actin and a C-terminal epitope of the TGF-β1 peptide (top panels). Supernatants were treated or not with acid and analyzed by ELISA to measure concentrations of total (latent + active) and active TGF-β1, respectively (bottom panels). Total TGF-β1 detected in the acid-treated samples corresponds to latent TGF-β1 because no active TGF-β1 was detected in the non-treated samples. Values represent means of duplicates + SD. <b>A</b>. Analysis of human T cell lines transduced or not with lentiviruses coding GARP or GFP. T cells were left resting (Rest) or stimulated for 24 hours with anti-CD3/CD28 antibodies (Stim) in serum-free medium. <b>B</b>. Analysis of stable clones of murine BW5147 T cells and 293 cells transiently transfected with <i>GARP</i> and WT or C33S mutant <i>TGFB1</i>. Untransfected BW5147 and 293 cells express low levels of endogenous TGF-β1 that are not detectable by WB in these conditions (not shown). By comparison to WT, transfection of mutant C33S results in increased production of total TGF-β1 (pro- + mature), as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076186#B49" target="_blank">49</a>].