a–d, Blocking activity of MAB3949 or Fab3949 of PAR2 activation by trypsin (a, c) or activating peptide SLIGRL (b, d) measured using FLIPR in human A549 cells. The IC50 of MAB3949 is 286 ± 143 nM for trypsin activation (n = 3) and 253 ± 36.7 nM for activating peptide (n = 2). Isotype controls (red curves) are mouse IgG2A clone NIP228 as full IgG or Fab fragment. Minor non-specific inhibition seen with high concentrations of full-length mouse IgG2A isotype control maybe related to IgG Fc domains as is not apparent with IgG2A Fab. Error bars represent s.e.m. e, f, Representative sensorgrams for interaction of Fab with full-length (e) and NΔ57 (f) PAR2-StaR variants. The mean Kd values determined from four replicate runs are 7.6 ± 1.9 nM and 11 ± 2.7 nM, respectively.