Pertussis toxin

The following reagents were purchased as indicated: recombinant human CCL19 and CCL21 (PeproTech, Rocky Hill, CT, USA), monoclonal anti-polyhistidine antibodies conjugated to HRP (Sigma, A7058-1VL, St. Louis, MO, USA), rabbit-anti-mRFP (PM005, MBL International, Woburn, MA, USA), anti-phospho-p44/42 MAPK (ERK1/2; pThr202/Tyr204; Cell Signaling Technology #4370, Danvers, MA, USA), anti-p44/42 MAPK (ERK1/2) Antibody (Cell Signaling Technology #9102), peroxidase conjugated AffiniPure goat-anti-rabbit IgG (Jackson Immunoreseach, West Grove, PA, USA), Fluo-3 AM (Molecular Probes, Eugene, OR, USA), Pertussis Toxin (Tocris, Bristol, UK).

For immunofluorescence studies, chicken anti-human/mouse RGS5 antibody was used (GW22900, Sigma Aldrich^®, Schnelldorf, Germany). For capillary electrophoresis, the anti-human/mouse RGS5 antibody (BYT-ORB6873, Biozol, Eching, Germany) was utilized to detect RGS5. The rabbit anti-human Ki67 antibody used for quantification of cell proliferation was purchased from Abcam (ab16667, Cambridge, UK). The mouse anti-human/mouse/rat RhoA (26C4) antibody (sc-418) and the mouse anti-recombinant His-6 antibody (sc-8036) were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). The mouse anti-human VCP antibody (ab11433, Cambridge, UK) was purchased from Abcam. Antibodies against total ERK1/2 (#4695) as well as phosphorylated ERK1/2 (#4370) were purchased from Cell Signaling Technology^® (Danvers, MA, USA). Pertussis Toxin (#3097) was purchased from Tocris (Bristol, UK) and YM-25490 (#257-00631) from Fujifilm Wako Chemicals Europe GmbH (Neuss, Germany).

The hFFAR1 low-expressing stable CHO-K1 cell line used in this study was purchased from Multispan, Inc (Cat # C1101-1A). Cells were maintained in DMEM/F-12 supplemented with 10% FBS, 1% 10000 U/mL penicillin and 100 μg/mL streptomycinand 10 μg/ml puromycin and incubated at 37°C with 5% CO2. The day before the assay, hFFAR1-expressing CHO-K1 cells were plated overnight in 384-well plates (4,000 cells per well) in complete media, with or without 100 ng/mL Pertussis Toxin (Tocris, Cat # 3097). The following day, the culture media was replaced with assay buffer containing HBSS with calcium and magnesium, 20 mM HEPES and 0.1% Fatty acid free BSA, pH 7.4. Compounds were then added and incubated with cells at 37°C for 90 min. Analytes were detected according to the manufacturer’s protocol (CisBio Ipone Tb kit, Cat # 62IPAPEC). Data presented are representative of at least three independent experiments performed in quadruplicate for each compound. Data are represented as averages ± S.D.

Female C57BL/6J mice (Harlan, UK) at 7 weeks of age received 500 μg of human RBP-1-20 peptide subcutaneously, emulsified in complete Freund's adjuvant (Sigma-Aldrich, UK) supplemented with 1.5 mg/ml Mycobacterium tuberculosis H37RA (Difco Laboratories, BD, Oxford, UK). Pertussis toxin (1.5 μg) was simultaneously administered into the peritoneal space (Tocris Bioscience, Bristol, UK). Imaging was performed 26 days later at the time point determined to be the disease peak in our facility using this protocol.