Anti crbn

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.

Proteins were extracted with lysis buffer (50 mM HEPES, 100 mM NaF, 10 mM EDTA, 50 mM Na pyrophosphate, 1% Triton at pH 7.4, and 10 mM Na Orthovanadate) for 15 min at 4 °C as previously reported [17 (link)]. The total protein samples (25 µg) were subjected to 9% SDS PAGE, transferred onto an activated polyvinylidene difluoride membrane (Immobilon P, Millipore, Denmark), and incubated overnight at 4 °C with primary antibodies followed by peroxidase-conjugated secondary antibodies. The signal was detected by Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, San Jose, CA, USA). Images were obtained using a densitometer (Bio-Rad Laboratories, Hercules, CA, USA) and resolved quantitatively. Each band level was standardized with respect to the β-actin control. Anti-asprosin (AdipoGen, Seoul, Korea), anti-PKA, anti-GLUT4, anti-SOD1, anti-SOD2, anti-β-actin antibodies (Santa Cruz, CA, USA), anti-CRBN (Sigma-Aldrich, Louis, MO, USA), anti-AMPK, anti-Akt, anti-p38MAPK, anti-TGF-β (Cell Signaling Technology, Beverly, MA, USA), anti-FNDC5, anti-PGC-1α, and anti-UCP3 antibody (Abcam, Cambridge, MA, USA) were used.