Figure 4.

Neutralization of secretory granules allows inhibition of tPA activity. (A) Bovine chromaffin cells were incubated for 90 min in a physiological saline solution with or without 25 mM NH₄Cl at 34°C to neutralize secretory granule pH. Cell lysates were resolved on a 10% SDS polyacrylamide gel containing casein (1 mg/ml) and plasminogen (10 µg/ml). SDS was removed by four washes in 2.5% Triton X-100 to allow renaturation of tPA. Gels (zymograms) were incubated in 100 mM Tris, pH 8.1, for 4 h at 37°C and then stained with Coomassie blue stain to visualize casein hydrolysis (inverted grayscale, in triplicate). (B) Gels were scanned, and band intensities were quantified in ImageJ. Mean ± SEM is shown. Student's t test resulted in a p-value of 0.0027.