Ab207442

Western blot analysis was performed as described [36 (link)]. Equal amounts of protein (15–20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane using Criterion XT Precast gels (Bio-Rad, USA). Membranes were blocked with 5% BSA for 2 h; then the corresponding antibodies GLUT4 (1 : 1000, #347063, Zen Bio), HK2 (1 : 1000, #200569, Zen Bio), PFKM (1 : 1000, #ab154804, Abcam), PKM (1 : 1000, #200667-1A7, Zen Bio), LDHA (1 : 500, #384822, Zen Bio), AMPKα (1 : 1000, #ab207442, Abcam), p-AMPKα (1 : 1000, #AP0116, ABclonal), mTOR (1 : 1000, #380411, Zen Bio), p-mTOR (1 : 1000, #5536, Cell signalling), 4EBP1 (1 : 500, #306002, Zen Bio), p-4EBP1 (1 : 500, #ab278686, Abcam), and Actin (1 : 2000, #200068-8F10, Zen Bio) were incubated overnight at 4°C; finally, blots were incubated with secondary antibodies at room temperature for 2 h. Bands were visualized with the gel imaging system (Syngene, UK) and analyzed with ImageJ analysis software (National Institutes of Health, https://rsb.info.nih.gov/ij/). The research met all the requirements of ARRIVE checklist and most of the requirements of CONSORT checklist if applicable (see supplemental files, named S1 ARRIVE Checklist and S2 CONSORT 2010 Checklist).

In total, 20 μg of protein of cytosolic fraction from each sample were mixed with loading buffer containing 0,125 M Tris (pH 6.8), 20% glycerol, 10% β-mercaptoethanol, 4% SDS, and 0.002% bromophenol blue, and heated at 50 °C for 5 min. Protein was electrophoresed on 10% SDS–PAGE gel using a miniprotean system (Bio-Rad, Madrid, Spain) with molecular weight standards (Bio-Rad). Protein transfer to nitrocellulose membranes was carried out in the iBlot^TM Dry Blotting System (Invitrogen, Madrid, Spain). Membranes were washed with PBS-Tween 20, blocked with PBS containing 5% skimmed milk, and then incubated with the primary antibodies at 4 °C overnight at 1:1000 dilution for anti-EAAT2 (Abcam, ab41621), 1:600 for anti-AMPK (Abcam, ab207442), 1:1000 for antiphosphorylated AMPK (Abcam, ab23875), and 1:2000 for anti-GAPDH (Abcam, ab8245). GAPDH was used as a gel loading control. After rinsing, the membranes were incubated with the corresponding secondary antibody (Bio-Rad, GAMPO 170-6516, GARPO 172-1019) at a dilution of 1:5000 in PBS containing 5% skimmed milk for 1 h. The antigen was visualized using the ECL chemiluminescence detection kit (Amersham, Madrid, Spain) in a G:Box chamber, and specific bands were quantified by densitometry using GeneTools software (Syngene).

Total proteins were isolated from cells using RIPA lysis buffer. After loading 25 μg cellular protein samples on SDS‐PAGE gels, proteins of the same weight from each group were separated and transferred to PVDF membranes. Subsequently, membranes were blocked with 5% skimmed milk for 1 h at room temperature and later incubated with primary antibodies overnight at 4°C. The main antibodies include rabbit anti‐human CD1E antibody (1: 1000, ab187157, Abcam), rabbit anti‐human STK11 antibody (1: 1000, ab138386, Abcam), rabbit anti‐human Bax antibody (1: 1000, ab32503, Abcam), rabbit anti‐human Bak antibody (1: 10000, ab32371, Abcam), rabbit anti‐human Bcl‐2 antibody (1: 1000, ab32124, Abcam), rabbit anti‐human AMPK antibody (1: 1000, ab207442, Abcam), rabbit anti‐human p‐AMPK antibody (1: 1000, ab133448, Abcam), and rabbit anti‐human β‐actin antibody (ab115777, Abcam). Following incubation with primary antibodies, they were incubated with secondary antibody IgG (1: 2000, ab97051, Abcam) for 1 h at room temperature. Protein bands were detected with enhanced chemiluminescence reagents (Merck Millipore). β‐actin was used as an internal reference.

JQJTT (190210) was provided by Longshunrong Pharmaceutical Factory of Tianjin Zhongxin Pharmaceutical Group Co., Ltd (Tianjin, China). PAL (CAS: 3486-67-7, purity >98%) was provided by Sichuan Vikqi Biotechnology Co., Ltd (Sichuan, China). FGF21 protein was obtained from Northeast Agricultural University. STZ and insulin were purchased from Sigma-Aldrich Co., Ltd (St. Louis, MO, United States). PD173074 was purchased from Selleck Chemicals (Houston, TX, United States). The glucose detection kit was obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Trizol and the cDNA reverse transcription kit were purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). SYBR Green qPCR Mix was provided by Shandong Sikejie Biotechnology Co., Ltd (Shandong, China). Primary antibodies targeting phosphorylated (p)-FGFR1 (ab173305), FRS2 (ab137458), AMPK (ab207442), p-AMPK (ab133448), and β-actin (ab8227) were purchased from Abcam (Cambridge, United Kingdom); those targeting p-FRS2 (YP0805), p-AKT (YT0185), and AKT (YP0006) were obtained from ImmunoWay (California,United States); and that targeting FGFR1 (9740T) was purchased from CST (Boston, MA, United States). Secondary antibodies were obtained from Absin Biotechnology Co., Ltd (Shanghai, China).

Radio Immunoprecipitation Assay (RIPA) solution with PMSF (1 mM) was used to isolate proteins from CLP-induced septic rat liver, kidney, and cardiac tissues. The initial protein concentrations were measured using the BCA protein assay kit. After the separation of proteins using 10% SDS-PAGE, PVDF membranes were used to transfer proteins electronically. After sealing in 5% BSA for 2 h, the membranes were incubated overnight at 4 °C with primary antibodies against p-AMPK (1:1000; ab133448; Abcam, Shanghai, China), AMPK (1:1000; ab207442; Abcam, Shanghai, China), p-mTOR (1:1000; ab109268; Abcam, Shanghai, China), mTOR (1:1000; ab32028; Abcam, Shanghai, China), and GAPDH (1:2500; ab9485; Abcam, Shanghai, China). The membranes were washed in triplicate with PBST and then the incubation was continued for 2 h using HRP-conjugated goat anti-rabbit IgG (1:1000; ab181662; Abcam, Shanghai, China) as the secondary antibody. The protein bands were visualized using the ECL detection reagent (Beyotime Institute of Biotechnology). Finally, protein concentrations were quantified using the ImageJ software.

Cell protein was prepared with the support of lysis buffer, and protein concentration was detected using the BCA kit. Then, 30 μg protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. Next, the membranes were sealed with 5% skimmed milk at ambient temperature for 1 h and then blotted with the following primary antibodies overnight at 4°C: WNK1 (ab137687, 1 : 2000, Abcam), LC3 I/II (ab128025, 1 : 1000, Abcam), Beclin 1 (ab210498, 1 : 1000, Abcam), P62 (ab91526, 1 : 1000, Abcam), AMPK (ab207442, 1 : 1000, Abcam), p-AMPK (ab23875, 1 : 1000, Abcam), and GAPDH (ab181602, 1 : 8000, Abcam). The membranes were blotted with the secondary antibodies for 1 h at ambient temperature. Protein bands were developed with enhanced chemiluminesence and then analysed using Image J software.