Biochemical Characterization of Cell Lines

HEK293 cells (ATCC number CRL-1573) and RAW264.7 cells (ATCC number TIB-71) were acquired from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), carboxymethylcellulose (CMC), 3-(4,5-dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyethylenimine (PEI), sodium dodecyl sulfate (SDS), ranitidine, dexamethasone (dexa), L-NAME, kaempferol, genistin, apigenin, lipopolysaccharide (LPS, E. coli 0111:B4), pam3csk4, and poly (I:C) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). RPMI 1640, DMEM, trypsin, PBS, and penicillin-streptomycin were received from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was acquired from Biotechnics Research, Inc. (Irvine, CA, USA). TRIzol reagent was purchased from MRCgene (Cincinnati, OH, USA). Primers used for semiquantitative RT-PCR and qPCR were purchased from Macrogen Inc. (Seoul, Korea). Phospho-specific or total-protein antibodies against p65, p50, IκBα, PI3K/p85, Src, c-Fos, c-Jun, JNK, ERK, p38, MKK4, MKK7, MEK1/2, TAK1, IRAK1, IRAK4, hemagglutinin (HA), and β-actin were acquired from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Constructs for signaling proteins HA-Src and HA-TAK1 were used as previously reported [24 (link),26 (link)].