Fig. 5.

Blocking anti-GARP mAb inhibits the integrin αVβ8-mediated TGF-β1 activation from GARP/TGF-β1 complexes. (A) The indicated Jurkat clones were stimulated (STIM) with anti-CD3/CD28 antibodies or not stimulated in the presence or absence of the indicated blocking antibodies. Cell lysates were collected after 24 h and analyzed by Western blot with antibodies against pSMAD2 or β-actin. mAbs used were as follows: anti-GARP, MHG-8 (mIgG1); anti-αV, 272–17E6 (mIgG1); and anti-β8, ADWA-11 (mIgG1). Images were captured with the Fusion Solo 4S station and quantified with Bio-1D software. (B) Quantification of pSMAD2:β-actin ratios measured by Western blot in three independent experiments performed as in A. Values represent mean ratios ± SD (**P < 0.01 and ***P < 0.001 vs. no antibody treatment by ANOVA followed by Tukey post hoc test).