Substance p

After expression, 1 × 10⁴ cells were incubated at 4 °C for 2 h in 200 μL binding buffer (50 mM Tris-HCl pH 7.4 (at 4 °C), 1 mM EDTA, 0.1% (w/v) BSA, 40 μg/mL bacitracin) containing [³H]-labelled ligand (NTR1 variants: 15 nM [3,11-tyrosyl-3,5-³H(N)]-neurotensin (Perkin Elmer); NK1R variants: 15 nM [leucyl-3,4,5-³H(N)]-substance P (Perkin Elmer); KOR1 variants: 15 nM [15,16-³H]-diprenorphine (Perkin Elmer)). Nonspecific binding was determined in the presence of a 1000-fold excess of unlabelled ligand (NTR1 variants: 10 μM neurotensin (8–13) (AnaSpec); NK1R variants: 15 μM substance P (AnaSpec); KOR1 variants: 15 μM diprenorphine (Tocris Bioscience)). After incubation, cells were filtered on MultiScreen filter plates (Merck Millipore) with a vacuum manifold, filters were washed four times with cold 50 mM Tris-HCl pH 7.4 (at 4 °C), transferred to Isoplate-96 scintillation plates (Perkin Elmer), dried at 65 °C for 2 h, and 200 μL Optiphase Supermix scintillation cocktail (Perkin Elmer) was added. Counting was performed on a 1450 MicroBeta Plus liquid scintillation counter (Wallac). Measured CPM values were normalized to the number of cells used in the assay and the nonspecific signal was subtracted from the total signal.

All cell culture reagents were obtained from Invitrogen (Gaithersburg, MD, USA). Amaxa transfection kit (Kit V) was obtained from Lonza (Gaithersburg, MD, USA). Q5 Site-Directed Mutagenesis Kit was from New England BioLabs (Ipswich, MA). Substance P (SP) was from AnaSpec (Fremont, CA, USA). Pertussis toxin (PTx) was from List Biological Laboratories (Campbell, CA, USA). YM-254890 was from Wako Chemicals (Richmond, VA, USA) and p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 acetoxymethyl ester was from Abcam (Cambridge, MA, USA). PE-conjugated anti-MRGPRX2 antibody was from BioLegend (San Diego, CA, USA). MRGPRX2 plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA).

All cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA); recombinant human stem cell factor (rhSCF), mouse interleukin-3 (mIL-3), and mouse stem cell factor (mSCF) were from PeproTech (Rocky Hill, NJ, USA); p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) was from Sigma-Aldrich (St. Louis, MO, USA) and Fura-2 acetoxymethyl ester was from Abcam (Cambridge, MA, USA). Substance P (SP) was from AnaSpec (Fremont, CA, USA). Phycoerythrin-conjugated anti-MRGPRX2, FITC-conjugated anti LAMP-1 and all other flow cytometry antibodies were from Biolegends (San Diego, CA, USA). Rabbit anti-Orai1, Orai2 and Orai3 antibodies from Alomone lab (Rockville, MD, USA), anti-ERK1/2, anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-phospho-Akt (Ser-473), anti-Akt, β-Actin and goat anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Danvers, MA, USA). SuperSignal West Pico Maximum Sensitivity Substrate was from Thermo Scientific (Rockford, IL, USA). Synta66 (3-fluoro-pyridine-4-carboxylic acid (2,5-dimethoxy-biphenyl-4-yl)-amide) was purchased from Calbiochem (San Diego, CA, USA). ELISA kits for mouse TNF-α, and human TNF-α, IL-8, CCL-3 were obtained from R&D system (Minneapolis, MN, USA). BCA Protein Assay Kit was obtained from Pierce Biotechnology (Rockford, IL, USA).