Figure 4.

Secretion of the p.K23_I24insIDK cationic trypsinogen mutant from transiently transfected HEK 293T cells. At 8, 24, 32 and 48 hours after transfection conditioned media were collected and 20 μL medium was supplemented with 0.1 M Tris-HCl (pH 8.0) and 1 mM CaCl₂ to 50 μL volume and trypsinogen was activated with 28 ng/mL human enteropeptidase for 1 h at 37 °C. Trypsin activity was then measured by adding 150 μL of the chromogenic substrate, N-benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide to 0.14 mM final concentration. Trypsin activities were expressed as percentage of the 48 h wild-type activity. The average of 2 independent transfection experiments is shown. For clarity, the error bars have been omitted; the standard error of the mean was within 15%. Inset: Eight hours after transfection 20 μL aliquots of conditioned media were electrophoresed on 15% SDS-polyacrylamide gels and analyzed by western blotting as described in Materials and Methods.