Fig. 6. TMPRSS13 silencing leads to increased protein levels of the tumor suppressor protease prostasin.

a Western blot analysis of TMPRSS13, matriptase, prostasin, and β-actin upon silencing with two non-overlapping RNA duplexes (siRNA 1 and siRNA 2) b Quantification of prostasin protein normalized to β-actin. c Invasion assays were performed in siRNA-treated HCC1937 cells 96 h post transfection with two non-overlapping synthetic RNA duplexes (siRNA-P1 and siRNA-P2) targeting prostasin and a %GC-matched RNA (in triplicates). Quantification of prostasin knockdown (KD) cells (red) and control cells (blue) invading through Matrigel (16 h) (upper panel) *p < 0.05, **p < 0.01 (one-way ANOVA, with Tukey’s post-hoc test for multiple comparisons). Western blot analysis to confirm prostasin silencing (96 h) (lower panel). Data are representative of three independent experiments. d Whole-cell protein lysates from HEK293T cells expressing empty expression vector (EV), expression vector with V5-tagged full-length human TMPRSS13 (TMPRSS13-V5) and EV, human full-length prostasin and EV, or TMPRSS13-V5 and prostasin were separated by SDS-PAGE under reducing conditions. Prostasin and β-actin were detected by western blotting. A lower molecular weight form of prostasin (cleaved prostasin?) is detected when co-transfected with TMPRSS13. e HEK293T cells expressing EV, prostasin/EV, or TMPRSS13-V5/prostasin, were immunoprecipitated with a mouse anti-V5 antibody and analyzed by western blotting (right panels) using the mouse anti-V5 antibody or a mouse anti-prostasin antibody. Whole-cell lysates were analyzed by western blotting to verity expression before precipitation (input, left panels). IgG H = IgG heavy chain detected by the secondary anti-mouse antibody. f HEK293T cells were transfected with full-length human wildtype prostasin (Pros-WT) and full-length human wildtype TMPRSS13 (T13-WT), T13-WT and zymogen locked prostasin (Pros-ZL), or Pros-WT and catalytically dead TMPRSS13 (T13-CD). Cells were treated with PI-PLC to release GPI-anchored prostasin 48 h post transfection. Released soluble prostasin was incubated with PN-1 (+) or buffer (−) for 1 h at 37 °C and samples were analyzed by SDS-PAGE under reducing conditions and western blotting using a mouse anti-prostasin antibody. The position of prostasin and prostasin-PN-1 complexes are indicated. A recombinant, soluble, truncated active form of purified prostasin protein (rProstasin) with or without PN-1 was included as positive control for complex formation. Lane 1: rProstasin; lane 2: rProstasin with addition of PN-1; lane 3: HEK293T cells expressing Pros-WT and T13-WT; lane 4: HEK293T cells expressing Pros-WT and T13-WT with addition of PN-1; lane 5: HEK293T cells expressing Pros-ZL and T13-WT; lane 6: HEK293T cells expressing Pros-ZL and T13-WT with addition of PN-1; lane 7: HEK293T cells expressing Pros-WT and T13-CD; lane 8: HEK293T cells expressing Pros-WT and T13-CD with addition of PN-1.