Fig. 3. GPR97 is a binding partner and allosteric activator of mPR3.
a Far-western blot analyses of the putative GPR97-ligand(s)(black arrowheads). The EMR2E-mFc protein was used as a negative control. Anti-CD11b and anti-GADPH Abs detected the corresponding protein markers of the membranous and cytoplasmic fractions, respectively. #, non-specific signals. Experiments were repeated 3 times with similar results. b FACS-based ligand-binding analysis of neutrophils pre-treated without or with phosphoinositide phospholipase C (PI-PLC). c Dot-plots of neutrophils double-stained with GPR97E-mFc and anti-CD177 or anti-PR3 mAb. d Dot-plots (FSC vs. surface CD177 levels) of neutrophils incubated with GPR97E-mFc. e IL-8 produced by unsorted and sorted neutrophils incubated with various reagents as indicated. n = 5 independent experiments. Cells treated with fMLF and LPS were included as positive controls. f Far-western blot analysis of specific GPR97-PR3 binding. Samples included total lysates of mock- and CD177-transfected HEK-293T cells as well as purified PR3. The arrowheads indicated the specific signals detected by the anti-PR3 mAb and the GPR97E-mFc probe. Experiments were repeated 3 times with similar results. g, h The FACS-based PR3-binding assay of unsorted g and FACS-sorted h HEK-293T cells expressing CD177 or GPR97. The black arrows in g indicated the specific PR3-binding. FACS-sorted GPR97high h and GPR97low l HEK-293T cells were examined. Numbers on top of the bars indicated the percentage of PR3-binding cell populations. i ELISA-like PR3-binding analyses. CD177-mFc and GPR56-mFc proteins were included as the positive and negative controls, respectively. n = 6 biologically independent samples. j, k ELISA analysis of IL-8 secreted by neutrophils treated without or with indicated protease inhibitors. n = 5 j, 4 k independent experiments. Data are presented as means ± SEM and p value was determined by two-way ANOVA in e, i–k. ns non-significant. l, m The ex vivo mPR3 enzymatic activity of neutrophils incubated in the absence or presence of mFc or GPR97-mFc protein probe without or with protease inhibitors as indicated. Source data are provided in the Source Data file.