Anti ampkα

K597, a DPP-4 inhibitor, and the Wako Labassay™ Glucose kit were from Wako Pure Chemical. Primary antibodies against GLUT1 and GLUT4, horseradish peroxidase-conjugated anti-goat, anti-rabbit and anti-mouse IgG antibodies and protein A/G plus-agarose were obtained from Santa Cruz Biotechnology Inc. Anti-AMPKα, anti-PI3K, anti-Akt and anti-insulin receptor (IR)β were from Cell Signaling Technology Inc. Anti-phosphotyrosine and anti-insulin receptor substrate (IRS)-1 were from Becton, Dickinson and Company. All other reagents used were of the highest grade available from commercial sources.

All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously^{26 (link)}. Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

SMI was purchased from CTQ Pharmaceutical Group Co. Ltd. (Hangzhou, China), the same batch as previous study (Yu et al., 2019 (link)). Cell culture supplies were purchased from Gibco (Grand Island, NY, USA). Anti-PI3K, anti-Akt, anti-Phospho-Akt (Ser473), anti-GSK-3β, anti-Phospho-GSK-3β (Ser9), anti-GAPDH, anti-AMPKα, anti-Phospho-AMPKα (Thr172), anti-Phospho-DRP1 (Ser616), anti-Phospho-DRP1 (Ser637), anti-Bax, anti-Bcl-2, anti-Caspase3, and anti-cleaved-Caspase3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-OPA1, anti-MFN2, and anti-FIS1 were purchased from Abcam (Cambridge, MA, UK). Anti-DRP1 and anti-MFN1 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). MitoSOX Red, MitoTracker Green, and MitoTracker Deep Red were purchased from Invitrogen (Eugene, USA).

The protein extraction method was according to our previous study^{19 ,22 (link)}. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 1% casein at room temperature for 1 h. Subsequently, the membrane incubated overnight at 4 °C with the primary antibodies against antiphospho-AMPKα (1: 1000, #2535, Cell Signaling Technology), antiAMPKα (1: 1000, #5831, Cell Signaling Technology), and antiβ-actin (1:3000, A1978, Millipore Sigma). After washing with TBST for four times, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using Tanon 4200SF system (Tanon Biotechnology, Shanghai, China) and quantified densitometry using Image J software (U.S. National Institutes of Health, Bethesda, MD).