Figure 3.

KLK6 induces loss of PAR1 and PAR2 from the surface of HT-29 cells and initiates calcium signaling via PAR2. (A) Immunofluorescence detection of PAR1 and PAR2 in HT-29 cells pre-treated with KLK6 (20 nmol/L), or with vehicle (control) for 15 min at 37 °C. Cells were fixed using 2% paraformaldehyde and immunostained with a PAR2 monoclonal antibody or PAR1 monoclonal antibody. Results are representative of two independent experiments (Magnification ×630). (B,C) KLK6 initiates intracellular Ca²⁺ mobilization. HT-29 cells were loaded for 60 min at 37 °C using Fura 2/AM. (B) HT-29 cells were challenged first with AP2 (SLIGKV-NH₂, 100 µmol/L)) and AP1 (TFFLLR-NH₂, 100 µmol/L). (C) Cells were first challenged with KLK6 (1 µmol/L) followed by a second challenge with activating peptide AP2 (100 µmol/L) and subsequent challenge with AP1 (100 µmol/L). Note the AP2 response showed a reduction in response, whereas cells were still responsive to AP1. Administration of compounds is indicated with the corresponding arrows.