Bca protein reagent

2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2-methyl-1,4-naphthoquinone (menadione), iron (II) sulfate (FeSO₄), hydrogen peroxide (H₂O₂), LPS (Escherichia coli serotype O111:B4), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), N-acetyl-L-cysteine (NAC), esterase, ConA, mitomycin C, dexamethasone, and BCA protein reagent were obtained from Sigma–Aldrich (St. Louis, MO, USA). The Griess reagent kit and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR, USA). Dulbecco’s Modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and streptomycin-penicillin were purchased from GIBCO (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The ONE-Glo^™ luciferase assay system and FuGENE^® HD transfection reagent were purchased from Promega (Madison, WI, USA). Hispolon and primary antibodies against phospho-IκBα, IκBα, NF-κB p65, JAK1, phospho-JAK1, STAT3, and transcription factor (TF) IIB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against phospho-STAT3 and α-tubulin and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

The total protein content of cell cultures was assessed using the bicinchoninic acid method (BCAProtein Reagent, Sigma), and the determination of absorbance at 570 nm was assessed using a spectrophotometric microplate reader (Infinite F200 Tecan). The amount of protein was measured by extrapolation from a standard curve using BSA as standard.