Bacterial Toxin Research Citations

Materials and Methods

Anthrax lethal factor, Product #169 and MAPKKide® Plus, Prod #532 are from List Biological Laboratories,
Inc. The 96-well, black, flat bottom, nonbinding plates used for the fluorescent plate assay were from
Corning (cat # 3991). The filtered adult bovine plasmas: Na-Citrate, cat #7310806; K2-EDTA, cat #7310807;
K3-EDTA, cat #7310808; Li-Heparin, cat #7310810 ; Na-Heparin, cat #7310811; and the filtered adult bovine
serum cat #7330800 were purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were made 1.25 mM
in DMSO. The substrate was then diluted 5-fold to 250 µM in assay buffer: 20 mM HEPES, pH 8.0 containing
0.1% Tween-20.

LF Activity Assay: LF was spiked into each plasma or serum sample and then each was diluted 1:5 using
assay buffer. The reaction was initiated by addition of 10 μM MAPKKide® Plus directly to the 1:5 diluted
plasma or serum, and the assays were run at 37°C using the kinetic mode of the plate reader with readings
at 1-minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 triplicates).

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

… A total of 31.7 nM PA alone (List Labs, no. 171E) or in the presence of 13.6 nM LFN, LFN-ACD, or LFN-RID was added to the media, and cells were incubated for 20 hours at 37C in the presence of 5% CO2 …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Reagents

Lethal factor (LF) was purchased from List Biological Laboratories (#169, recombinant from B. anthracis)

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Protein expression and purification

Upon addition of double molar excess of trypsin inhibitor (Sigma Aldrich), PA7 was further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare). Lyophilized LF (List Biological Lab. Inc., Lot#1692A1B) were resuspended in water according to the manufacturer’s manual and mixed with PA7 in a molar ration of 10:1. …

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Reagents

… Lethal Toxin was from List Biological Laboratories. …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

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

Chemicals

… PA63 protein was prepared from PA83 using furin (53) or purchased from List Biological Laboratories (Campbell, CA) in the purified form. …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

… 1:1,000 for antiEF and antiLF sera, and antiPA antibody (List Labs, Campbell, California). …

Chemicals and Reagents:

… Recombinant LF was from List Biological Laboratories (Campbell, California). …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

… For intoxication of cells, media was replaced over 105 cells previously seeded in a 12-well plate. 31.7 nM PA alone (List Labs, #171E) or …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Reagents and antibodies

… For cell culture experiments, Val-boroPro was resuspended in DMSO containing 0.1% TFA to prevent compound cyclization. Anthrax Lethal Factor (Catalog #172B) and PA (Catalog #171E) were purchased from List Labs, …

LDH cytotoxicity assays

For LDH release experiments involving primary macrophages, mBMDM and rBMDM were isolated from the femurs and tibias of 79-week-old male and female mice and 712-week-old male rats. 110⁷ cells were plated on 10cm Petri dishes. mBMDMs were differentiated in DMEM with 10% FBS and 20% L Cell medium for 6 days and rBMDMs in 30% L Cell medium for 79 days. Macrophages were scraped and reseeded at 0.2510⁶ cells/well in 12-well plates in Opti-MEM and incubated overnight before treatment. Cells were then treated as indicated with compounds or LT. …

In vitro and in cell LF cleavage assays

HEK 293T cells were seeded at 0.510⁶ cells/well in six-well plates and transiently transfected with 2g of the indicated plasmids using FuGENE HD. For the in vitro assay, lysates were harvested after 48h and normalized to 1mg/mL using the DC Protein Assay kit (Bio-Rad). These lysates were then incubated with LF (1g/mL) for the indicated time periods at 25C quenched by boiling with 2SDSPAGE sample buffer at the indicated times, separated by SDSPAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-Cor). For the in cell assay, after 48h after transfection cells were treated with LT (1g/mL, 6h) before lysates were harvested for immunoblotting as described above.

• Product #172B – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis