Determination of cathepsin enzyme activity in HIV-1–treated MDDCs. Day-6 immature MDDCs were exposed to HIV-1_BaL (MOI 10) or mock treated for 48 hours and then lysed in a low pH buffer. (A) Affinity labeling of active cysteine proteases. Cell lysates were incubated with the activity-based probe for cysteine cathepsins DCG-0N. Labeled cathepsins then detected by Western blot. Lanes: (1) Mock MDDCs, leupeptin, (2) mock MDDCs, (3) HIV-1_BaL-treated MDDCs, (4) boiled HIV-1_BaL–treated MDDCs, (5) monocyte organelles, (6) macrophage cell lysate. Vertical lines denote a section of the Western blot that was included with a lower exposure time. (B) Fluorogenic substrate measurement of cathepsin activity. The combined catalytic activities of cathepsins X, B, L, and S were determined fluorometrically by cleavage of the common synthetic substrate Z-Phe-Arg-7-amido-4-methylcoumarin. Specific catalytic activities of cathepsins S and C were measured using the substrates Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH₂ and NH₂-Gly-Arg-AMC, respectively. Samples were measured in triplicate.