Human PAR4 expression and purification. Full-length human PAR4 was expressed and purified from Sf9 cells. The protein was purified by FLAG antibody affinity chromatography followed by size exclusion chromatography. (A) A size exclusion chromatogram was obtained from purifications. Human PAR4 with a C-terminal 1D4 tag eluted in the main peak from fractions 14 to 20 (0.5 mL per fraction). The other small peaks contain tobacco etch virus for cutting the b562 RIL (BRIL) sequence from PAR4, the BRIL sequence, and the FLAG peptide for affinity chromatography. (B) Using both Coomassie staining (left) and immunoblotting with 1D4 antibody (right), the purity of the purified human PAR4 was confirmed. Purified PAR4 is a 37-kDa band on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an extra glycosylated PAR4 band. The thrombin cleavage removed 2.9 kDa from the protein, and the activated PAR4 is at 34 kDa. (C) A G-protein assay measured the relative fluorescence change of tryptophan in the PAR4 and G-protein complex to confirm that the purified PAR4 is functional.