Journal: PLoS ONE

Article Title: Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent

doi: 10.1371/journal.pone.0007913

Figure Lengend Snippet: (A) C57BL/6 Nlrp1b(129S1) BMDMs were pre-treated with varying concentrations of z-FA-FMK, CA074Me, or CA074 for 4 hours, followed by addition of 400 ng/mL of PA and 300 ng/mL of LF (LT), or no treatment (NT), for an additional 3.5 hours. Cytotoxicity was measured using ATP-lite. BMDMs from three C57BL/6 Nlrp1b(129S1) were used with each condition tested in quadruplicate. Error bars represent standard deviation. (B) RAW 264.7 cells were pre-treated with varying concentrations of z-FA-FMK, CA074Me, or CA074 for 4 hours, followed by addition of 400 ng/mL of PA and 300 ng/mL of LF (LT), or no treatment (NT), for an additional 3.5 hours. Cytotoxicity was measured using ATP-lite. The data presented here are representative of three or more independent experiments. Each point represents the mean of triplicate or quadruplicate samples from a single experiment, with error bars representing standard error. (C) RAW 264.7 cells were pre-treated with 100 µM z-FA-FMK or 50 µM CA074Me for 4 hours followed by LT (400 ng/mL PA and 300 ng/mL LF; +) or no toxin (−) for 2.5 hours. Cell lysates were subjected to western blot analysis and probed with an antibody that recognizes the N-terminus of MEK2. (D) Specificity of the ctsB probe Ak-EVD-AMK was determined by treating RAW 264.7 or NIH 3T3 cells with or without high concentration (5 or 10 µM) of probe. Cycloaddition assays were performed on Ak-EVD-AMK labeled cellular lysates with azido-rhodamine, and Ak-EVD-AMK labeled proteins were analyzed by in-gel fluorescence. The pro-form of ctsB is 43 kDa, whereas active ctsB is seen as either 31 kDa or 25 kDa. (E) In-gel fluorescence showing electrophoretically separated proteins from RAW 264.7 cells treated with 400 ng/mL of PA and 300 ng/mL of LF and/or, low dose (625 nM) Ak-EVD-AMK. Under these conditions, cytosolic ctsB is preferentially labeled. Cycloaddition was preformed as in (D). The gel was then stained with coomassie to indicate equal sample loading.

Article Snippet: CA074 (N-1475) and CA074Me (N-1660) were purchased from Bachem (Torrance, CA).

Techniques: Standard Deviation, Western Blot, Concentration Assay, Labeling, Fluorescence, Staining