FIGURE 5.

In vitro proteolytic processing of substrate candidates by other KLKs. KLK5, -7, -8, -13, or -14 was tested (all produced in-house as recombinant proteins). Briefly, 100 ng of recombinant MDK (A), CYR61 (B), and 2.5 μg of TNC (C) were incubated with active KLK5, -7, -8, -13, or -14 for 3 h, respectively. KLK5 (2.5 nm), KLK7 (3.3 nm), KLK8 (3.1 nm), KLK13 (3.1 nm), or KLK14 (3.2 nm) was used to digest midkine and CYR61. For TNC, KLK5 (62.5 nm), KLK8 (83 nm), KLK8 (83 nm), KLK13 (83 nm), or KLK14 (83 nm) was added into the reaction. D, KLK5, -7, -8, -13, or -14 alone was incubated at 37 °C for 3 h or overnight, respectively. The reaction was stopped, and the cleaved products were then separated by 4–12% SDS-PAGE followed by silver staining or Coomassie Blue staining. The arrows indicate the cleaved products. For more comments see “Results.”