Ab2907

Recombinant human CRT was obtained from Novus Biochemicals. Bovine liver CRT was purified according to (70 (link)) with modification. The procedure consisted of (NH₄)₂SO₄ precipitation of bovine liver homogenates, followed by ion-exchange chromatography with diethylaminoethyl cellulose and Concanavalin A-Sepharose chromatography. Aliquots of the eluted fractions from Concanavalin A-Sepharose were assayed by SDS-PAGE and stained with Coomassie blue and Stains-All (2 (link)) and confirmed with an anti-CRT mAb (Abcam # ab2907, Sigma (HPA002242) and Abcam # ab92516, US Biologicals (C1036)). CRT used in these studies was >90% pure. Expression and purification of yeast WT recombinant human CRT and the frameshift mutants were as described (52 ). Human recombinant single-chain tPA was obtained from Genentech and further purified by chromatography on benzamidine-sepharose. Recombinant pro-uPA was obtained from Axxora. S100A10 was purified according to (95 (link)). Human Glu-plasminogen and Lys-plasminogen were obtained from Enzyme Research Laboratories. Unless specified otherwise, Glu-plasminogen was used in all experiments. Mitoxantrone (Cayman Chemical Company) and oxaliplatin (Tocris) were purchased from Cedarlane Labs.

Western blotting was employed to determine the total expression of CRT. Briefly, cells were lysed in radioimmunoprecipitation assay buffer with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) augmented with 1 mM PMSF. The prepared lysates were separated (25 μg) on 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked (Odyssey blocking buffer-Licor, Lincoln) and incubated with anti-CRT primary antibody (Abcam Ab2907 and Ab92516), anti-tubulin (Sigma, Canada), and anti-GAPDH (Santa Cruz Biotechnology Inc) primary antibodies overnight at 4 °C, and with IR-conjugated secondary antibody, DyLight 680/800 (Thermo Fisher Scientific) for 1 h. The stained membranes were scanned using the Odyssey scanning system (Licor).