Citations

Assessment of light chain protease activity (VAMP-2):

The protease activities of BoNT/B1 (List Laboratories, US), rBoNT/B1 and rBoNT/B1_(S201P) against VAMP-2 were assessed using the BoTest (Biosentinel, Wisconsin, US) cell-free assay. BoNT/B1, rBoNT/B1 and rBoNT/B1_(S201P) were diluted to 1.39 nM in BoTest Reaction Buffer (50 mM HEPES-NaOH, 5 mM NaCl, 10 M ZnCl₂, 0.1% Tween-20, 0.1 mg/mL BSA, pH 7.1). BoNTs were reduced at room temperature (20 2C) for 30 minutes by addition of 5 mM DTT to allow maximum catalytic activity in the assay. BoTest Reporter (VAMP-2 (3394) flanked by N-terminal cyan fluorescent protein (CFP) and C-terminal yellow fluorescent protein (YFP) in 50 mM HEPES-NaOH, 10 mM NaCl, 15% glycerol) at a final concentration of 200 nM was combined with reduced BoNT/B1, rBoNT/B1 or rBoNT/B1_(S201P) (final concentration 0.5 pM1.25 nM) in black Maxisorp plates (Nunc) in a final assay volume of 100 µL/well. The plates were sealed, wrapped in aluminium foil to prevent degradation of the light-sensitive substrate and incubated at 30°C for 18 h. After incubation, fluorescence emission at 485/20 nm and 528/20 nm following excitation at 440/40 nm was measured using a BioTek Synergy HT plate reader.

Induction and assessment of EAE:

Foxp3^CreERT2 and Foxp3^CreERT2DOCK8^fl/fl mice were injected s.c. with 100 µg of MOG_35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (Difco) supplemented with 4 mg/ml Mycobacterium tuberculosis extract H37Ra (Difco). Additionally, mice received 200 ng pertussis toxin (List Biological Laboratories) i.p. at day 0 and 2. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… EdU, sections were first incubated with goat anti-CTB antibody (1:5000, List Biological Laboratories, Campbell, CA, USA) …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

… 10 ng/ml cholera-toxin (List Biological Laboratories, Inc., Campbell, California), …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Bacterial culture, infection procedures, OMV isolation, and treatments:

For treatment of BMDMs, OMVs or LPS (Ultra-pure LPS from Salmonella enterica serovar Minnesota R595; List Biological Laboratories) was diluted in Opti-MEM (Gibco) and added directly to cells. …

• Product 434 – ULTRA PURE LPS from Salmonella minnesota R595 (Re)

Induction of EAE

For active EAE induction, mice were immunized s.c. with MOG₃₅₅₅/CFA at the tail base on day 0. Pertussis toxin (List Biological Laboratories) was injected intraperitoneally (i.p.) on day 0 and day 2. … The recipient mice were immunized with MOG₃₅₅₅/IFA followed by two PTX injections on the day of immunization and 2 days later. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

cAMP Assay:

LS174T cells, HEK-EP2 cells, and HEK-DP cells were cultured in 6-well plates and, prior to experiments, culture media were switched to Opti-MEM (Thermo Scientific) containing the corresponding antibiotics as stated above for 16h. In the case of LS174T cells, cells were treated for 25min with 0.1mg/mL isobutylmethyl-xanthine (Sigma, St. Louis, MO, U.S.A.) following to a pretreatment with the indicated concentrations of MW-03 for 16h. Cells were then treated with vehicle or the indicated concentrations of PGE₂ (Cayman Chemical, Ann Arbor, MI, U.S.A.) at 37C for 1h or 0.01µg/mL cholera toxin (CTX, List Biological Laboratories, Campbell, CA, U.S.A.) for 4h. …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

… was coated with purified goat F(ab’) 2 anti-mouse IgM + IgG + IgA antibody (200 ng/well in 100 µl of PBS; SouthernBiotech, Birmingham, AL), attenuated cholera toxin (CT) (200 ng/well in 100 µl of PBS; List Biological Laboratories …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

The Chronic EAE Model:

To evaluate AIS integrity in an inflammatory environment, we induced the chronic
model of experimental autoimmune encephalomyelitis (EAE) as previously described
(DeVries et al., 2012; Secor McVoy et al., 2015; Dupree et al., 2015). … Mice were also injected intraperitoneal (i.p.) on the same day with 300 ng Pertussis toxin (PT) (List Biological Labs, Campbell, California) in 200 µL phosphate buffered saline (PBS) with a booster PT injection 48 hours later. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

 … MOG emulsion was transferred to 1 ml Luer-Lok syringes (BD Falcon) with 27G inch needles. Pertussis toxin was diluted in UltraPure (2 ng/l; List Biologicals Laboratories, Campbell, CA) and transferred to syringes as described above. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)