Chemiluminescence kit

The total protein concentration was measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). In brief, 30 μg of proteins were loaded into each lane of sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and then transferred to polyvinylidene fluoride membranes (Pall Corporation, Beijing, China), which were incubated with primary Abs against IRAK4, phospho-IRAK4, P38, phospho-P38, JNK, phospho-JNK, TRIF, IRF3, phospho-IRF3, NF-κB p65, phospho-NF-κB p65, IKKα, IKKβ, phospho-IKKα/β, IkBα, and phospho-IkBα (Cell Signaling Technology, Inc., Beverly, MA, USA; all diluted to 1:1000), as well as GAPDH (1:5000) and PSGL-1 (Santa Cruz Biotechnology, Inc.; 1:1000). Anti-rabbit or mouse horseradish peroxidase-linked Ab (ZSGB-BIO Technology Co., Ltd., Beijing, China; 1:8000) was used as the secondary Ab. Detection was performed using a chemiluminescence kit (Advansta, Inc., Menlo Park, CA, USA). Densitometry of proteins was analyzed with Gel-Pro software (Media Cybernetics).

All chemicals used were of analytical grade and purchased from Sigma Aldrich, and SRL, Mumbai (India). ¹H NMR spectra were recorded on a Agilent (400 MHz) spectrometer in CDCl₃ solvent, using TMS as an internal standard, ¹³C NMR spectra were recorded on a Agilent (100 MHz) spectrometer. Mass spectra were determined on PE Sciex API3000 ESI-MS, elemental analyses were carried out using an Elemental Vario Cube CHNS rapid analyzer. Progress of the reaction was monitored by TLC pre-coated silica gel plates.
HepG2 cell line was initially purchased from ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum, 1 mM l-glutamine, 1 mM sodium pyruvate, antibiotic, and antimycotic agent. GAPDH, lamin B, and p65 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-IκBα (Ser 32), IκBα, phospho-p65 (Ser 536) was purchased from Cell Signaling Technology (Beverly, MA, USA). Nuclear extraction and NF-κB DNA binding kits were purchased from Active motif (USA). Blocking buffer was purchased from Nacalai Tesque (Kyoto, Japan). Chemiluminescence kit was purchased from Advansta (CA, USA). The small interfering RNA (siRNA) for NF-κB and scrambled control was obtained from Santa Cruz Biotechnology. Caspase-Glo 3/7 assay kit and luciferase substrate was purchased from promega (WI, USA).

Total protein of TSG-6 mRNA-transfected cAT-MSCs was extracted using Pro-Prep protein extraction solution (iNtRON Biotechnology). The concentration of extracted protein was quantified using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). We separated 20 μg of protein by SDS-PAGE, which was then transferred to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA). The membranes were blocked with 5% non-fat dry milk and exposed to primary antibodies against TSG-6 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-actin (1:1,000, Santa Cruz Biotechnology) at 4°C for 24 h. Membranes were washed and then incubated with secondary antibodies at room temperature for 1 h. We detected the immunoreactive bands of the proteins using a chemiluminescence kit (Advansta, San Jose, CA, USA); the protein expression levels were normalized to that of β-actin.