P erk1 2

CNE2 cells with different concentrations of luteolin (0 μM, 20 μM, and 40 μM) and CIS (10 μM) were incubated for 36 h, total protein was extracted and quantified, and 50 μg of protein was used for western blot analysis. The appropriate concentration of separating gel and stacking gel was chosen depending upon the molecular weight of the target protein. The samples were then loaded, onto the SDS-PAGE gel, electrophoresed and the resolved proteins were transferred to a transfer membrane (PVDF), and blocked. Diluted β-actin (Cell Signaling Technology, CST, MA, USA), p-ERK1/2 (CST), ERK1/2 (CST), AKT (CST), PI3K (CST), PCNA (Bioss, Woburn, MA, USA), and XIAP (CST) at a dilution ratio of 1 : 1,000 were incubated with the PVDF membrane overnight at 4°C, then wash three times with TBST, for 10 min each. Then, the corresponding diluted goat anti-mouse or goat anti-rabbit secondary antibody was added, and incubated with the PVDF membrane at room temperature for 2 h, washed with TBST three times for 10 min and then scanned on an ODYSSEY CLx Infrared Imager (LICOR, Lincoln, NE, USA) to detect the signal intensities of the immunoreactive bands. Using β-actin as an internal reference, the comparative protein expression levels could be calculated, the experiment was performed in triplicate.

ISL and dexamethasone (DEX) were provided by Macleans (Shanghai, China). LPS from Escherichia coli 055:B5 serotype, purity ≥ 99%, item number: L8880) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide] were provided by Solarbio (Beijing, China). The fluorescence quantification kits were purchased from Takara (Beijing, China). Primary antibodies were obtained commercially, including p-p65, p-IκBα, p65, IκBα, p-ERK1/2 and ERK1/2 (Bioss, Beijing, China) as well as JNK, p38, p-JNK and p-p38 (Wanleibio, Shenyang, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were supplied by Gibco (Suzhou, China). The ELISA and NF-κB Activation Nuclear Transport Test Kits were available from SinoBestBio (Shanghai, China) and Beyotime (Shanghai, China), respectively. Tris-buffered saline plus Tween 20 (TBST) was obtained from Solarbio (Shanghai, China).