Ab227069

Animal studies were conducted with approval by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) (protocol A00002844-17) according to protocols that we have described in detail previously (51 (link)). Female Nod/SCID mice (6–8 weeks old) received an intraperitoneal injection with either JHOC9 (2 × 10⁶) or JHOC5 (5 × 10⁵) tumor cells injected into the lower right quadrant of the abdomen. JHOC9 cohorts were euthanized by CO₂ asphyxiation at 15 weeks and JHOC5 cohorts at 7 weeks due to tumor growth kinetics and moribund endpoints. Ascitic fluid was retrieved from the abdominal cavity and centrifuged to pellet the cellular component. The supernatant was collected and stored at −20 °C until use. Clarified supernatant (100 μl) was incubated with 16 μM activity-based probe for 1 h at 37 °C to achieve protein labeling. Samples were then evaluated by Western blot for uPA (Invitrogen MON-U-16-02, dilution 1:1000; goat anti-mouse secondary Thermo Scientific #31432, dilution 1:1000) or tPA (Abcam #Ab227069, dilution 1:1000; donkey anti rabbit secondary GE Healthcare NA934V, dilution 1:1000). Evidence of probe labeling was assessed using HRP-conjugated streptavidin (Cell Signaling #3999, incubation 1:7000, 1 h at RT). Membranes were washed three times and developed with Clarity Western ECL (BioRad) for 5 min before exposure to film.

JHOC9 cells transduced with PLAU-targeted or nontarget control lentiviral shRNA, or JHOC5 cells transduced with PLAT-targeted or nontarget control lentiviral shRNA, were seeded and cultured in 10 cm plates as detailed above for ABPP in-situ labeling. After 24 h in 3 ml of serum-free phenol red–free media at near confluency, cultures were labeled with 5 μM activity-based probe at 37 °C for 1 h as described. Supernatant was clarified and cell debris removed. Samples were then evaluated by Western blot for uPA (GeneTex GTX10046, dilution 1:1000; donkey anti-rabbit secondary GE Healthcare NA934V, dilution 1:3000) or tPA (Abcam #Ab227069, dilution 1:1000; donkey anti-rabbit secondary GE Healthcare NA934V, dilution 1:1000). Probe labeling was assessed using HRP-conjugated streptavidin (Cell Signaling #3999, incubation 1:7000, 1 h at RT). Membranes were washed three times and developed with Clarity Western ECL (BioRad) for 5 min before exposure to film.