Elution solvent (acetonitrile), step gradients (0, 20%, 32%, 50%, 65%, or 90% for 0 minutes, 10 minutes, 40 minutes, 55 minutes, 70 minutes, or 80 minutes, 1.6 mL/minute, 203 nm), and a phenomenex gemini C18 ODS (250 mm × 4.6 mm, 5 μm) column were used. Based on these conditions, the contents of ginsenosides from PPD-SF were calculated with the peak area curve of standard ginsenosides. To evaluate cytokine mRNA expression levels, RAW264.7 cells pretreated with PPD-SF (0–400 μg/mL) for GSK-3 signaling pathway 30 minutes were incubated with LPS (1 μg/mL) for 6 hours. Total RNA was isolated with TRIzol Reagent (Gibco BRL) according to the manufacturer’s instructions and stored at −70°C

until use. The mRNA was quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with SYBR

Premix Ex Taq, according to the manufacturer’s instructions (Takara, Shiga, selleck screening library Japan), using a real-time thermal cycler (Bio-Rad, Hercules, CA, USA), as reported previously [23] and [24]. Results were expressed as the ratio of the optical density relative to glyceraldehyde 3-phosphate dehydrogenase. The primers used (Bioneer, Seoul, Korea) are described in Table 1. HEK293 cells (1 × 106 cells/mL) were transfected with 1 μg of plasmid containing β-galactosidase and NF-κB-Luc, AP-1-Luc, or IRF-3-Luc in the presence or absence of PMA, or overexpressed adaptor molecules (TRIF or MyD88) using the polyethylenimine (PEI) method in 12-well Thiamine-diphosphate kinase plates. The cells were treated with PPD-SF for 12 hours prior to termination. Luciferase assays were performed using the Luciferase Assay System (Promega, Madison, WI, USA), as previously reported [24] and [25]. Stomach tissues or RAW264.7 cells (5 × 106 cells/mL) were washed three times in cold phosphate-buffered saline with 1mM sodium orthovanadate, and then

lysed using a sonicator (Thermo Fisher Scientific, Waltham, MA, USA) or a Tissuemizer (Qiagen, Germantown, MD, USA) in lysis buffer [26] for 30 minutes with rotation at 4°C. Lysates were clarified by centrifugation at 16,000 × g for 10 minutes at 4°C and stored at −20°C until use. Nuclear fractions were prepared with RAW264.7 cell-derived lysates in a three-step procedure [27]. After treatment, cells were collected with a rubber policeman, washed with 1 × phosphate-buffered saline, and lysed in 500 μL lysis buffer [28] on ice for 4 minutes. Lysates were centrifuged at 19,326 × g for 1 minute in a microcentrifuge. The pellet (nuclear fraction) was washed once in washing buffer (lysis buffer without Nonidet P-40) and then treated with extraction buffer (lysis buffer containing 500mM KCl and 10% glycerol). The nuclei/extraction buffer mixture was frozen at −80°C, thawed on ice, and centrifuged at 19,326 × g for 5 minutes. The supernatant was collected as a nuclear extract. Soluble cell lysates (30 μg/lane) were immunoblotted.