Erk1 2

Anti-RxRxxS*/T*(#10001S), RSK1 (#9333S), RSK2 (#5528S), p-eIF4B (#3591S), eIF4B (#3592S) antibodies were ordered from Cell Signaling Technology. HRP anti-HA (#901519), HRP anti-Flag (#637311) antibodies were purchased from BioLegend. pRSK1 (Thr359/Ser363) (#AP0539), pRSK1 (Ser380) (#AP1147), RSK3 (#A16305), ERK1/2 (#A10613), and SUMO1 (#A19121) were purchased from ABclonal. Anti-RTA (ORF50) monoclonal mouse antibody was given by Dr. Ke Lan (Wuhan University, China) [34 (link)]. Anti-ORF65 monoclonal mouse antibody was given by Dr. Shou-Jiang Gao (University of Pittsburgh, USA) [35 (link)]. Monoclonal antibodies against ORF45 and ORF52 and polyclonal antibodies against K3, K8, and ORF55 were described previously [36 (link)–38 (link)]. EZview Red ANTI-FLAG M2 Affinity Gel (#F2426) and sodium butyrate (#B5887) were ordered from Sigma, Doxycycline (#S4163) was ordered from Selleck, Glutathione Sepharose 4B (#17-0756-01) were ordered from GE Healthcare. ClonExpress II One Step Cloning Kit (#C122-01) and HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (#R223-01) were purchased from Vazyme Biotech. Lipofectamine 3000 (#3000015) were purchased from Thermo Fisher Scientific.

In our study, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS solution), 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N,N′,N′-tetramethylethylenediamine (TEMED), acrylamide/bisacrylamide, ammonium persulfate (APS), ascorbic acid (vitamin C), bovine serum albumin (BSA), ethanol (96%), glycine, methanol, Ponceau S, resazurin sodium salt (RES), sodium dodecyl sulfate (SDS), Trolox, trypsin-EDTA solution, Tris-HCl, Tris-Base, and Tween-20 were purchased from Merck KGaA (Darmstadt, Germany). The PVDF membrane with 0.45 µm-pore size and primary antibodies against SOD1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies against GAPDH, NF-κB, IκBα, SRC, GLUT4, PCNA, PGC-1α, PPARγ, p-ERK1/2, and ERK1/2 were purchased from ABClonal (Woburn, MA, USA). The primary antibodies against NLRP3, anti-mouse- and anti-rabbit-HRP-conjugated antibodies were purchased from ThermoFisher (Waltham, MA, USA). Potassium persulfate (Warchem, Zakręt, Poland), antibiotics (Penicillin-Streptomycin, Life Technologies, Bleiswijk, The Netherlands), DMEM (Dulbecco’s Modification of Eagle’s Medium, Biological Industries, Beit Haemek, Israel), FBS (Fetal Bovine Serum, Biological Industries, Genos, Lodz, Poland), and phosphate buffered saline (PBS, pH 7.00 ± 0.05, ChemPur, Piekary Ślaskie, Poland) were also used during the analyses.

Strong RIPA lysis buffer (GenStar, Nanjing, China) was used to lyse cells, bicinchoninic acid (BCA) was used to quantify proteins (GenStar, Nanjing, China). Then, 10% SDS-PAGE and PVDF membrane (Millipore, Burlington, MA, USA) were performed to separate and transfer protein. Primary antibodies, including DAXX (Abways, Beijing, China), ERK1/2 (ABclonal, Wuhan, China), p-ERK (Cell Signaling Technology, Connors Farm, MA, USA), mTor (Cell Signaling Technology, USA), and actin (Cell Signaling Technology, USA) were incubated with membrane at 4 °C for more than 8 h. Then, secondary antibody (1:500) was incubated with membrane for 1 h at room temperature. Finally, the blots were visualized by Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA). ImageJ was used to analyze the results.

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).