mAb-RC3 inhibits thrombin cleavage of both Ala120 and Thr120 PAR4 variants. Thrombin cleavage of PAR4 was assessed in HEK293T cells transiently transfected with either Ala120-PAR4 or Thr120-PAR4 variants with an N-terminal FLAG tag. (A) Cells were stimulated with increasing concentrations of thrombin and receptor cleavage measured as a loss of FLAG-epitope by flow cytometry. (B) Preincubation of transfected cells with mAb-RC3 before thrombin stimulation provided equivalent concentration-dependent inhibition of thrombin cleavage of either PAR4 variant. Note near-complete inhibition of thrombin cleavage with higher concentrations of mAb-RC3. Data are mean ± standard error of the mean of n = 3 individual experiments each performed in duplicate.