Typhoon flatbed laser scanner

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.

Cells were lysed with PBS 0.1% Triton X-100 and total protein concentration was determined by BCA assay. Protein (80 µg) was acidified by adding 10× citrate buffer (final concentration: 50 mM citrate, pH 5.5, 0.5% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonic acid, 0.1% Triton X-100, 4 mM dithiothreitol) or PBS containing 0.1% Triton X-100). BMV109 (pan cysteine cathepsins probe, 1 μM) was added to the acidified cell lysate at 37˚C as previously described^{43 (link)}. Proteins were resolved by reducing SDS-PAGE and in-gel Cy5 fluorescence was detected using a Typhoon flatbed laser scanner (GE Healthcare). Protease activity (band intensity) was measured by densitometry using ImageJ. Values were normalized to actin (band intensity). Statistical analyses were performed using GraphPad Prism 8.