Anthrax Edema Factor (EF), Recombinant from B. anthracis

Chemicals and reagents

… Toxins (product numbers) purchased from List Biological Laboratories were: EF (178A), diphtheria toxin (150), Clostridium septicum alpha toxin (116L), C. perfringens epsilon toxin (126A), C. difficile toxins A (152C), B (155B), and binary toxin subunit A(157A), Pasteurella toxin (156), pertussis toxin (181), botulinum neurotoxins A (612A) and B (622A) (heavy chains), and cholera toxin (100B). …

• Product #178A - Anthrax Edema Factor (EF), Recombinant from B. anthracis
• Product #150 - Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae
• Product #152C - Toxin A from Clostridium difficile
• Product #155 - Toxin B from Clostridium difficile
• Product #156 - Pasteurella multocida Toxin
• Product #181 - Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #612A - Botulinum Neurotoxin Type A Heavy Chain, Recombinant, Binding Domain
• Product #622A - Botulinum Neurotoxin Type B Heavy Chain, Recombinant, Binding Domain
• Product #100B - Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Toxins:

...  Recombinant EF and PA from Bacillus anthracis were purchased from List Biological Laboratories, Inc., Campbell, CA. ...

Signal Transduction Experiments:

Zymosan particles were opsonized by incubating the particles with 50% human serum for 30 min at 37 C. The particles were then washed and dissolved in DMEM with FCS before use for treatment of cells (100 g of zymosan particles per 10⁶ cells). To investigate the effects of toxin-provoked cAMP intoxication on opsonophagocytic signaling, 3 10⁶ THP-1 cells were preincubated with CyaA (150 ng/ml; 0.85 nM) for 30 min or ET (625 ng/ml; 7.04 nM EF + 2500 ng/ml; 30.24 nM PA) for 6 h, respectively (based on cAMP ELISA experiments to have similar cAMP intoxication levels, cf. Figure 1A,B). The cells were then treated with 300 g of serum opsonized or unopsonized zymosan as positive and negative controls for 30 min, respectively.

• Product #171E - Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A - Anthrax Edema Factor (EF), Recombinant from B. anthracis

Lep-vesicle-recycling endosome-exocyst complex inhibition tests:

The cell monolayers were treated with 35 nM edema factor or lethal factor plus 70 nM protective antigen (EF + PA or LF + PA) of anthrax toxin (List Biological Laboratories, USA), the inhibitor (EF + PA) of Rab11 and inhibitor (LF + PA) of Sec15 to block Rab11-Sec15 binding, for 24 hr at 37C (Guichard et al., 2010), and then infected with L. interrogans strain Lai at MOI100 for 8 hr. The subsequent steps and detection of Lep-vesicle-Rab11-Sec15/Sec3 co-localization were the same as above. ...

• Product #171 - Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A - Anthrax Edema Factor (EF), Recombinant from B. anthracis
• Product #169 - Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Efferocytosis Assay:

Efferocytosis was assessed by flow cytometry [68]. Briefly, 5 10⁵/well monocyte-derived human M2(IL-4+IL-10+Dex) macrophages or M2(Dex) macrophages were preincubated with 10 nM recombinant PA (List Biological Laboratories, Campbell, CA, USA), 10 nM recombinant EF (List Biological Laboratories), or various concentrations of PA + EF (ET) for 4 h at 37 C in 5% CO₂ atmosphere. Fluorescently-labeled apoptotic human PMN were preincubated with 10% serum, Protein S, or MFGE8 for 1 h unless otherwise noted and then added to the macrophages, resulting in a macrophage:PMN ratio of 1:5. After 1 h at 37 C in 5% CO₂ atmosphere, the percentage macrophages containing intracellular (eFluor 670⁺) but not surface-bound (surface CD66b) PMN was assessed by flow cytometry.

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A (Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Reagents:

... PA (catalog number 171E) and EF (catalog number 178a) were purchased from List Biological Laboratories (Campbell, CA).

ChIP-Seq and data processing:

PBMC were plated at 2 million cells per ml in RPMI containing 10% FBS, 100 units/ml penicillin, and 100g/ml streptomycin. These cells were then treated with 10nM ET (10nM EF and 10nM PA) or 500M 6MB-cAMP for 4.5h and then isolated by centrifugation followed by snap freezing in liquid N₂ as recommended by Active Motif. For each condition, 60 million PBMC were prepared from 3 donors (20 million PBMC per donor). ChIP was performed by Active Motif with antibodies validated for ChIP against CREB (Cell Signaling Tech; catalog number 9197) or against C/EBP (Santa Cruz Biotechnology, catalog number sc-150). After ChIP DNA libraries were constructed, 75-nt sequence reads were generated by Illumina sequencing.

• Product #171E - Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A - Anthrax Edema Factor (EF), Recombinant from B. anthracis

ELISPOT:

Peripheral blood mononuclear cells (PBMCs) were prepared and washed in RPMI medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and L-glutamine. ...The antigen, recombinant Protective Antigen (rPA), LF or EF (List Biological Laboratories Campbell, Calif.) was added at a final concentration of 12.5 g/ml for PA or 25 g/ml for either LF or EF. The plates were incubated overnight at 37C in a 5% CO₂, humidified atmosphere. ...

Quantitative anti-PA IgG ELISA:

Wells of 96-well microtiter plates (Immulon 2HB; Dynex Technologies, Chantilly, Va.) were coated by adding of 100 l rPA (List Biological Laboratories Campbell, Calif.) diluted to 1 g/ml in PBS. Plates were incubated overnight at 4C. ...

Lethal toxin neutralization assay (TNA):

...Lethal toxin (LeTx; 100 ng of rPA/ml and 50 ng of LF/ml, final concentrations) was prepared in the same medium and pre-incubated with the sample dilutions in a humidified incubator set at 37C, 5% CO2 for 1 h. LeTx was prepared using PA obtained from List Biological Laboratories ...

End-point ELISA for LF and EF:

Wells of 96-well microtitre plates (Immulon 2HB) were coated with either LF or EF (List Biological Laboratories) at 1 g/ mL in PBS (100 L per well). ...

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

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

 Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Recombinant proteins and reagents:

Protective antigen (PA) and edema factor (EF) were purchased from List Biological. ...

Cell seeding and incubation with toxins:  

For toxin treatment, patterned and nonpatterned peritoneal macrophages and BMDMs were incubated with PA at 1 g/ml and EF at 100 ng/ml for 6 or 16 h at 37C in a 5% CO₂ atmosphere. For some experiments, cells were incubated with PA alone at 1 g/ml.

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

In vitro protease assays:

Purified InhA1 was mixed with the substrate in protease buffer (50 mM Tris, 100 mM NaCl, 1 mM CaCl₂, 1 mM MgSO₄ [pH 7.8]) and incubated at 37C for set time points. Reaction mixtures were then placed on ice, and EDTA was added to a final concentration of 50 mM to stop the reaction. SDS buffer (2) was added, and samples were boiled for 5 min, separated by SDS-PAGE, and analyzed by Western blotting with substrate-specific antibodies. Recombinant PA (rPA), rLF, and rSodA-1 from B. anthracis were obtained from BEI Resources (ATCC, Manassas, VA). rEF was obtained from List Biological Laboratories (Campbell, CA). rNpr599 was purified as described above.

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Human cytokine antibody array assay: 

After separation by centrifugation, WBCs were washed twice with complete RPMI 1640 medium, and the cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin. WBCs (5 10⁶ to 10 10⁶ cells per ml) were treated with ET (25 pmol PA plus 25 pmol EF), LT (25 pmol PA plus 25 pmol LF), ...

The following materials were used in this study. B. anthracis EF, LF, and PA were purchased from List Biological Laboratories (Campbell, CA).

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

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).