Brilliant blue g

Berbamine (BBM), Mitomycin C, Pluronic F-127, poly (ethylene glycol)-10,000 (PEG – 10,000), Rhodamine 123, Tween-80, propidium iodide (PI), 6-coumarin, protease inhibitor cocktail, sodium dodecyl sulphate (SDS), glycine, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), D-α-Tocopherol poly (ethylene glycol) 1000 succinate (TPGS), bradford reagent, triethyl amine (TEA), crystal violet, gelatin, brilliant blue G, p-coumaric acid and luminol were procured from Sigma-Aldrich (St. Louis, MO). Glycerylmonooleate (GMO) was purchased from Eastman (Memphis, TN). Sodium chloride was procured from MP biomedical (Cedex, France). Acetonitrile and methanol were obtained from Spectrochem, Pvt. Ltd. (Mumbai, India). Dimethyl sulphoxide, ammonium acetate and acetic acid were obtained from Merck, India Pvt. Ltd. (Mumbai, India). Tris base was purchased from Promega (Promega Corporation, Madison, Wisconsin). All other chemicals used were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification.

Protein content was measured using the Bradford method [51 (link)]. The procedure is based on formation of a complex between dye (brilliant Blue G, Sigma-Aldrich, Dorset, UK) and the protein present in the sample, and absorbance is read at 595 nm using a spectrophotometer. 50 µL filtered crude enzyme extract was added to 1.5 mL of concentrated dye reagent, vortexed and allowed to stand for 20 min before taking the absorbance reading. Bovine serum albumin (BSA) (Sigma-Aldrich, Dorset, UK) was used to construct a standard curve, and the protein concentration of sample was calculated from the standard curve obtained. Protein content was used to calculate the specific activity of myrosinase enzymes (U/mg protein).

All chemicals used in this study were of the highest purity >99.9% and quality. Indomethacin, ibuprofen, KCl, Na₂SeO₃, latex beads, carboxylate-modified polystyrene, fluorescent red, bovine serum albumin, brilliant blue G, p-nitrophenyl butyrate, 2-nitrophenyl-B-D-galactopyranoside, 1-chloro-2,4-dinitrobenzene, L-glutathione reduced, sodium phosphate dibasic were purchased from Sigma-Aldrich. CaCl₂·2H₂O, MgSO₄·7H₂O, NaHCO₃, HCl, p-nitrophenyl phosphate, boric acid, ammonium acetate, NaOH, methanol, and DMSO were purchased from Fisher Scientific.

P2 receptor antagonists: Pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), MRS2211, MRS2179, MRS2159, A438079, Brilliant Blue G, ATP-2′, 3′-dialdehyde (OxATP), niflumix acid, carbenoxolone disodium salt, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, United States). NF449, NF023, and Evans Blue tetrasodium salt were purchased from Tocris Bioscience (Abingdon, United Kingdom). Suramin hexasodium salt was purchased from Aladdin (Shanghai, China). Anti-His monoclonal antibody was purchased from EARTHOX Life Sciences (Millbrae, CA, United States). Anti-ETX monoclonal antibody was developed previously by colleagues in our laboratory. HRP-coupled goat anti-mouse IgG antibody was purchased from TransGen Biotech (Beijing, China). The ATP assay kit was purchased from Calbiochem-Merck (Darmstadt, Germany). MTS (3-(4, 5-dimethylthiazol-2-yl) -5(3-carboxymethoxyphenyl) -2-(4-sulfopheny)-2H-tetrazolium, inner salt) was purchased from Promega Corporation (Madison, WI, United States).

Uric acid, lipopolysaccharide (LPS; from Escherichia coli 0111:B4), Brilliant Blue G, pyrrolidinedithiocarbamate (PTDC), and HEPES were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wortmannin was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Acetyl-YVAD-chloromethylketone and TAK242 were purchased from Calbiochem (Rockland, MA, USA). Pam3CSK4 was purchased from Tocris (Bristol, UK). Antibodies against phosphorylated-Akt (p-Akt), Akt, caspase-1 P10, and caspase-1 P20 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against ABCG2, PDZK1, Na/K ATPase, Lamin A/C, GAPDH, β-actin, TLR2, TLR4, MYD88, P2X7, ASC, and nuclear factor-κB (NF-κB) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Penicillin/streptomycin and TRIzol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).