Erk1 2

Cells were harvested with RIPA lysis buffer containing 0.5% NP-40, 150 mM NaCl, 1 mM EDTA (pH 8.0), 50 mM Tris-HCl (pH 8.0), and protease inhibitors. Protein extracts were quantified and separated by electrophoresis on a 15% SDS–PAGE gel. Primary antibodies including HIF1α (1:1000, NB100–105, Novus, Littleton, CO, USA), HIF2α (1:500, NB100–132, Novus), GAPDH (1:10,000, SC32233, Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-tubulin (1:10,000, bs1699, Bioworld, Dublin, OH, USA), GSK3β (1:1000, Cell Signaling Technology, 9332, Danvers, MA, USA), p-GSK3β (1:1000, Cell Signaling Technology, 9336), ERK1/2 (1:1000, bs1112, Bioworld), and p-ERK1/2 (1:1000, bs4621, Bioworld) were used. The secondary antibodies used were goat anti-mouse (1:10,000, 31,439, Pierce, Rockford, IL, USA) and goat anti-rabbit (1:10,000, Sigma-Aldrich).

Cells were stimulated with E₂, G1, or TAM for 15 min with or without G15, AG, U0126, and WM pretreatment. Western blotting was then performed as described previously (Liu et al. 2010 (link)). Briefly, cell lysates were harvested in a cell lysis buffer (Boster, Wuhan, China), dissolved in 9% SDS–PAGE buffer, and subjected to western blotting using primary detection antibodies against total or phosphorylated ERK1/2 (diluted 1:1000; BioWorld, St Louis Park, MN, USA) and GPER. Membranes were incubated overnight at 4 °C before incubation with the appropriate HRP-conjugated secondary antibodies. Immunodetection was conducted using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). Optimal density was analyzed using Image J Software, and results were expressed as fold change relative to the control.

The following reagents were used: rHMGB1 (Cat No. #1690-HMB-050, R&D Systems, Minneapolis, MN, USA), G1 (Cat. No. 3577/10, TOCRIS, Bioscience, Bristol, UK), G15 (Cat. No. 3678/10, TOCRIS, Bioscience, Bristol, UK). E2, TAM, were obtained from Sigma–Aldrich (St. Louis, MO, USA). U0126, and the LY294002 were purchased from Millipore (Temecula, CA, USA). E2 was dissolved in ethanol and other drugs were solubilized in dimethyl sulfoxide (DMSO; Sigma–Aldrich). The following antibodies were used: anti-HMGB1 neutralizing antibody (Cat No. H00003146-M08, R&D systems, Minneapolis, MN, USA); p-ERK1/2 (Cat No. AP0484P, diluted 1:1000), ERK1/2 (Cat No. BS90472, diluted 1:1000), Akt (Cat No. BS6473, diluted 1:1000) were purchased from Bioworld (St Louis Park, MN, USA). p-Akt (Cat No. 4060, Cell signaling Technology, USA, diluted 1:1000), LC3B (Cat No. 2775 Cell Signaling Technology, diluted 1:1000), P62 (Cat No. 5114, Cell Signaling Technology, diluted 1:1000); Beclin1(Cat No. 210498, diluted 1:1000), GPR30 (Cat No. ab39142, diluted 1:250), HMGB1 (Cat No. ab18256, diluted 1:500), Bcl-2 (Cat No.32124, diluted 1:1000), BAX (Cat No. 182734, diluted 1:1000) were purchased from Abcam (Cambridge, MA, USA), and β-actin (Cat No. TA-09, diluted 1:1000) from Zhongshan Golden Bridge (Beijing, China).

GE (Figure 7A) and SIN (Figure 7B) (purity > 98.0%) were provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ, IL-17, IL-4 and TGF-β1 were supplied from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Rabbit mAb JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, and β-actin were obtained from Bioworld Technology Inc. (Nanjing, China). HRP-conjugated goat anti-rabbit antibodies were purchased from ZSGB-BIO (Beijing, China). JNK inhibitor (SP600125), ERK inhibitor (PD98059), and p38 inhibitor (SB203580) were obtained from Calbiochem-Novabio chem Corp. (San Diego, CA, USA). Lipopolysaccharide (LPS), Cell counting kit-8 (CCK8) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were offered from Sigma Chemical Co. (St. Louis, MO, USA); Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo Scientific Co. (Waltham, MA, USA). All other chemicals used in this study were of analytical grade and were obtained from commercial sources.

The muscle lysates were prepared as described by Wang et al. [23] (link), and subjected to SDS-PAGE gel electrophoresis and western blot analysis using appropriate antibodies: mTOR, FoxO4, p-mTOR (Ser2448), p-FoxO4 (Ser193) and p-ERK1/2 (Thr202/Tyr204) were purchased from Cell Signaling Technology, Inc. (Danvers, MA), and ERK1/2 from Bioworld Technology, Inc. (St. Louis Park, MN). After washing, membranes were incubated with a secondary antibody (Rockland Immunochemicals, Gilbertsville, PA). The bands were visualized by infrared fluorescence using the Odyssey Imaging System (LI-COR) and quantified by Odyssey infrared imaging system software.