P jnk

RAW264.7 cells (1 × 10⁶ cells/well) were treated with the same way of ELISA or inoculated in dishes with a diameter of 6 cm overnight and stimulated with CPS or LPS for the indicated times, respectively. Then, cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) on ice for 30 min, and proteins were extracted and quantified by BCA Kit (Beyotime, Jiangsu, China). 40 µg of protein was separated via 8 or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto Polyvinylidene Fluoride (PVDF) membranes (Millipore, CA, USA). The membranes were blocked with 5% Bovine Serum Albumin (BSA) or skim milk at room temperature for 1 h and then incubated with primary antibodies specific for iNOS, ERK1/2, pERK1/2, P38, and JNK1/2/3 (ABclonal, Wuhan, China), and COX2, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), p-P38, and pJNK (Bioworld Technology, Inc, MN, USA) at 4°C overnight. The membranes were then incubated for an additional 1 h with goat antirabbit lgG/ Horseradish Peroxidase (HRP) (Bioss, Beijing, China). Then, the membrane was developed using HyperSignal ECL Plus kit (4A Biotech, Beijing, China) for imaging with ChemiScope 3000 mini (Clinx, Shanghai, China).

Afatinib, adriamycin and cisplatinum were purchased from Meilun Biology Technology Co., Ltd (Dalian, China). Paclitaxel was obtained from Tianfeng Technology Co., Ltd (Xi'an, China). Rhodamine 123, MTT, verapamil, lipopolysaccharide (LPS), PDTC, LY294002 and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MQ, USA).
The monoclonal antibody against ABCB1 was obtained from Santa Cruz Biotechnology Inc. (CA, USA). Antibodies against iκBα, p-iκBα, p65 and Lamin B1 were purchased from Epitomics, Inc. (California, USA). Antibodies against EGFR, p-EGFR, HER-2, p-HER-2, AKT, p-AKT, p38, p-p38, ERK1/2, p-ERK1/2, JNK, p-JNK and GAPDH were obtained from Bioworld Technology, Inc. (Minnesota, USA). The HRP-conjugated goat anti-rabbit secondary antibody and the CY3-conjugated goat anti-rabbit secondary antibody were purchased from Abcam Inc. (Cambridge, MA, USA).

RAW264.7 cells (1 × 10⁶/well) were cultured in a 6-cell plate overnight and then pretreated with E9OAEE (6.25, 12.5, 25, 50 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h or indicated time, respectively. Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) on ice for 30 min, and the supernatant was collected. Proteins were quantified using Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China), and equal amounts of protein (40 µg) were separated via 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto PVDF membranes (Millipore, CA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies specific for iNOS (ABclonal, Wuhan, China), GAPDH (Bioworld Technology, Inc, MN, USA), COX2 (Bioworld Technology, Inc, MN, USA), JNK1/2/3 (ABclonal, Wuhan, China), p-JNK (Bioworld Technology, Inc, MN, USA), P38 (ABclonal, Wuhan, China), p-P38 (Bioworld Technology, Inc, MN, USA), ERK1/2 (ABclonal, Wuhan, China), and p-ERK1/2 (ABclonal, Wuhan, China). The membrane was then incubated for an additional 60 min with a goat anti-rabbit lgG/HRP (Bioss, Beiing, China). Then, the membrane was developed using Super ECL Plus kit (US Everbright Inc, Suzhou, China) for imaging with ChemiScope 3000 mini (Clinx, Shanghai, China).