Ikkα β

After 12 or 24 h of treatment, RSC-364 cells and lymphocytes were harvested. Cells were lysed using RIPA Lysis Buffer containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Protein samples (2 mg/ml) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto the polyvinylidene difluoride membranes (Sigma, St. Louis, MO, USA). The membranes were blocked with 5% skim milk and incubated overnight with the following primary antibodies at 4°C: T-bet, GATA-3, RORγt, TRAF2, IKKα/β, phospho-IKKα/β, and NF-κB p50 for lymphocytes; TRAF2, MyD88, IKKα/β, phospho-IKKα/β, NF-κB p50, JAK1, STAT3, and phospho-STAT3 for RSC-364 cells (1 : 1000, Cell Signaling Technology, MA, USA). The membranes were then incubated with horseradish peroxidase- (HRP-) conjugated IgG secondary antibody (1 : 3000, Abcam, Cambridge, MA, USA) at room temperature for 2 h. All immunoreactive proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Three replicates of each experiment were performed. The densitometry values were normalized to GAPDH and quantified using Image-Pro Plus version 4.0 (Media Cybernetics Inc., Rockville, MD, USA).

RAW264.7 cells and colon tissues were homogenized, and protein levels were quantified using BCA reagent (Beyotime, Shanghai, China). Nuclear and cytoplasmic extractions were performed as instructed by the manufacturer (Beyotime, Shanghai, China). Samples were loaded and electrophoresed in 10–12% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk diluted in TBS-T for 2 h followed by incubation with primary antibodies. The following primary antibodies were used in this experiment: iNOS, COX-2, phospho-IKKα/β, IKKα/β, phospho-IκBα, IκBα, phospho-ERK ½, ERK ½, phospho-p38, p38, p65, phospho-c-Jun, c-Jun, c-Fos, β-tubulin, Lamin A/C, phospho-JNK, JNK, IRAK4, and β-actin at 1:1,000 dilution (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG at 1:2,500 dilution.

Methanol extraction of P. attenuatum (Pa-ME) was purchased from the Plant Extract Bank in the Plant Diversity Research Center (Daejeon, Republic of Korea; http://extract.kribb.re.kr, e-mail: mplantext@kribb.re.kr). RAW264.7 cells, a transformed macrophage cell line derived from the BALB/c mouse (ATCC number TIB-71), were purchased from ATCC (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), L-N^G-nitroarginine methyl ester (L-NAME), indomethacin, lipopolysaccharide (LPS, Escherichia coli 0111:B4), pam3CSK, and (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Poly I:C was obtained from Calbiochem (La Jolla, CA). The enzyme immune assay (EIA) kits used to quantitate the levels of PGE₂ were purchased from Amersham (Little Chalfont, Buckinghamshire, UK). Specific PCR primers for iNOS, TNF-α, COX-2, and GAPDH were synthesized from Bioneer Inc. (Daejeon, Republic of Korea). Antibodies that specify phosphorylated and total forms of p65, p50, c-Jun, c-Fos, Lamin A/C, IκBα, IKKα/β, AKT, Src, Syk, ERK, p38, JNK, IRAK1, IRAK4, TAK1, MKK3/6, and β-actin were obtained from Cell Signaling (Beverly, MA, USA).