Chemicals and Reagents:
All bacterial toxins were purchased from List Biological Laboratories (Campbell, CA, USA). …
LF FRET Drug Screen and Data Analysis:
For screening JHCCL drugs in 96-well plates, the reaction volume was 250l per well, containing 20mM HEPES pH 7.2, 5M MAPKKide conjugated with DABCYL and FITC (List Biological Laboratories, Inc), and 33M of JHCCL compound. Reactions were initiated by adding anthrax LF…
Cell Culture, Toxins Treatments, and Cell Viability Assays:
RAW264.7 mouse macrophage cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Corning) and 100g/ml penicillin and streptomycin. …Cells were treated with drug hits for 1hour at 37C 5% CO2. RAW264.7 cells were challenged with anthrax toxins that include LF and native 83kDa PA (for 3hours), LF and PA in the 63kDa form (for 6hours), P. aeruginosa exotoxin A (for 12hours), or Cholera toxin (for 12hours), which were also pre-treated with identical drug concentrations, such that the final toxins concentrations were 0.5, 0.5, 2.0, and 4.0g/ml respectively. …
Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae