Fig. 7.
aPC signaling requires PAR2 in addition to PAR3 on T-cells. a Following knockdown of PAR2 in human primary pan T-cells (T) using two different shRNAs (shRNA1 and shRNA2) the inhibitory effect of aPC on T-cells after stimulation with αCD3 and αCD28 antibodies is lost. Scrambled shRNA (shRNAsc) had no effect. Bar graph summarizing results from five repeat experiments using five biological disjunct donors, each in triplicates. b Preincubation of T-cells from C57BL/6 wild type (B6T(aPC)) mice, but not of C57BL/6 PAR2-deficient (PAR2−/−(aPC)) mice, with aPC and subsequent stimulation with plate-bound αCD3 and αCD28 antibodies, abrogates aPC’s inhibitory effect on T-cell reactivity. c Preincubation of human pan T-cells with aPC reduces ERK1/2 and p38 activity after 96 h MLR as reflected by reduced phosphorylation of these proteins (normalized to the corresponding total protein levels) as determined by immunoblot; bar graph reflecting mean results ± SEM (top) and representative immunoblots (bottom) are shown. d Exemplary immunoblot showing interaction of PAR2 and PAR3 in human Tregs analysed using immunoprecipitation for PAR2 followed by immunoblotting of PAR3 and vice versa. Immunoprecipitation using IgG was used as negative control. Immunoblot of PAR2 and PAR3 was done as a input control. Mean value ± SEM (a, b), results of at least three repeat experiments each with three biological disjunct donors (a, c) or from five different mice (b). *P < 0.05, **P < 0.01, NS non significant (a, b ANOVA; c t-test)