Tri reagent solution

Cell pellets were resuspended in 2 ml of TRI Reagent solution (Molecular Research Center) and the suspension was split into two 1 ml aliquots that were placed into individual 2 mL microcentrifuge tubes. The suspensions were incubated at 60°C for 10 min, and centrifuged (10 min, 16,000 x g at 4°C). Each supernatant was transferred to a 2 ml tube, 200 μl of chloroform (Sigma) was added, the solution was mixed for 15 sec, incubated for 5 min at 25°C, and centrifuged (15 min, 16,000 x g at 4°C). 500 μl of each aqueous phase was added to a 2 ml tube containing 500 μl of 90% ethanol. Samples were mixed and transferred to RNeasy mini columns (Qiagen). RNA isolation was carried out according to the manufacturer’s instructions (Qiagen). RNA samples were eluted in 170 μl of nuclease-free water and treated with 20 units of Turbo DNase (Ambion) for 1 h at 37°C. 1 ml of TRI Reagent solution (Molecular Research Center) and 200 μl of chloroform (Sigma) were added, samples were mixed, incubated for 5 min at room temperature, and centrifuged (10 min, 16,000 x g at 4°C). 600 μl of the aqueous phase was transferred to a 2 ml tube containing 1.4 ml of 100% ethanol. Samples were incubated for ~12 h at −20°C, precipitated, washed twice with 1 ml of 75% ethanol, and resuspended in 50 μl of nuclease-free water.

RAW264.7 cells (ATCC TIB-71) and HEK293T cells (ATCC CRL-1573) were purchased from the American Type Culture Collection (Rockville, MD, USA). Penicillin, streptomycin, fetal bovine serum (FBS), Roswell Park Memorial Institute 1640 (RPMI 1640) medium, Dulbecco’s modified Eagle’s medium (DMEM), and Opti-MEM™ Reduced Serum Medium were obtained from GIBCO (Grand Island, NY, USA). Lipopolysaccharide (LPS), Poly (I:C), Pam3CSK4, MTT, polyethylenimine (PEI), dimethyl sulfoxide (DMSO), and dexamethasone were bought from Sigma Chemical Co. (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was obtained from Samchun Pure Chemical Co. (Gyeonggi-do, Republic of Korea). TRI Reagent^® solution was purchased from Molecular Research Center, Inc. (Cincinnati, OH, USA). The cDNA synthesis kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primers for semiquantitative RT-PCR and quantitative rea-time PCR were synthesized from Macrogen (Seoul, Korea), and PCR premix was purchased from Bio-D Inc. (Seoul, Korea). The ELISA kit for measuring TNF-α protein level was from R&D Systems (Minneapolis, MN, USA). Total and phospho-p50, p65, IκBα, AKT, p85, and Src targeting antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against β-actin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).