Zebrafish matriptase cleaves Par2b. (A) Proposed Par2b activation mechanism. Cleavage at R28/M29 unmasks tethered peptide ligand. (B) AP-Par2b cleavage reporter. Cleavage releases AP to conditioned medium. (C–E) HEK293 cells were cotransfected with empty pcDNA3.1 or the same vector directing expression of the Par2b cleavage reporter AP-Par2b, AP-Par2b carrying a R28A/M29P mutation to ablate the predicted activating cleavage site (AP-Par2b csm), or zebrafish matriptase (St14a). AP activity released into the conditioned medium was measured luminometrically. (C) AP released by trypsin treatment (4 nM for 10 min) to measure releasable AP. (D) AP released during a 45-min incubation to assess the effect of matriptase (St14a) expression. (E) AP activity in detergent extracts of cells expressing AP-Par2b or AP-Par2bmut was measured as an index of total expression of the reporter constructs. Data shown are mean ± SEM of three biological replicates, with AP activity expressed as arbitrary luminescence units. This experiment was done three times with similar results.