Af2058

Cells were washed with PBS and lysed by freezing in citrate buffer (50 mM citrate, pH 5.5, 0.5% CHAPS, 0.1% Triton X-100, 4 mM DTT). Supernatants were cleared by centrifugation. Total protein concentration was determined by BCA assay (Pierce). Activity-based probes [BMV109 (0.1 μM) or LE28 (1 μM)] were added to lysates from a 100x stock, and proteins were incubated at 37°C for 45 minutes [51 (link)]. Reaction was stopped by addition of 4x sample buffer (40% glycerol, 200 mM Tris-Cl, pH 6.8, 0.04% bromophenol blue, 5% beta-mercaptoethanol) followed by boiling for five minutes. Total protein (30–50 μg) was resolved by SDS-PAGE (15%). Gels were then scanned for Cy5 fluorescence using a Typhoon flatbed laser scanner (GE Healthcare). Where required, gels were transferred to nitrocellulose membranes using a Trans-Blot Turbo Transfer System (BioRad) and subject to standard western blotting protocols. Antibodies: goat anti-cathepsin X (1:1000; R&D AF1033), goat anti-cathepsin B (1:1000; R&D AF965), goat anti-cathepsin L (1:1000; R&D AF1515), sheep anti-legumain (1:1000; R&D AF2058), goat anti-cathepsin S (1:500; Abcam 18822), goat anti-cathepsin K (1:500; Abcam 19027). Tissues labeled with BMV109 in vivo were lysed by sonication in citrate buffer, and then equal protein was analyzed by fluorescent SDS-PAGE as above.