Anti insulin receptor β

The reagents used for different parts of this work were obtained from the indicated suppliers as follows: RNAProtect cell reagent (QIAGEN, 76526), NucleoSpin RNA isolation kit (Macherey Nagel, 740955), Quanti-Tect reverse transcription kit (QIAGEN, 205313) and Taqman probes (Thermo Fisher): Ins2 (Mm00731595_gH), Mer (Mm00434920_m1), rhodopsin (Mm01184405_m1) and Ins1 (Mm01950294_s1). Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V). We also used Halt Phosphatase inhibitor (Thermo Fisher, 1862495), Halt Protease inhibitor (Thermo Fisher, 1862209), ultrasensitive mouse insulin ELISA (Mercodia, 10-1249-01), mouse C-peptide ELISA (CrystalChem, 90050), rat/mouse C-peptide 2 ELISA (Millipore, EZRMCP2), glucose Glo Assay (Promega, J602), lactate Glo Assay (Promega, J5021) and eBioscience Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher, 00-5523-00).

Protein lysates were prepared from isolated tissue using RIPA lysis buffer with added Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Equal amounts of protein lysate were loaded in TGX Precast gels (Bio-Rad) or in-house-made 7% polyacrylamide gels, then subjected to SDS–PAGE. Protein was transferred onto PVDF membranes using the Trans Blot Turbo transfer system (Bio-Rad). After transfer, blots were blocked for 1–3 h in 5% dry milk in TBST at room temperature, then probed using anti-phospho insulin receptor β (Tyr1150/1151) (19H7) (1:1,000 dilution, Cell Signaling, 3024), anti-insulin receptor β (1:5,000 dilution, Novus Biologicals, NBP2 12793), anti-Glut4 (1:1,000 dilution, Alomone Labs AGT, 024), anti-β actin HRP (1:15,000 dilution, Sigma, A3854) and Amersham ECL anti-rabbit IgG HRP (1:5,000 dilution, GE Healthcare, NA934V). In some experiments, primary and secondary antibodies were diluted in SignalBoost Immunoreaction Enhancer kit solutions (Millipore Sigma). Blots were exposed using Western Lightning Plus ECL kit (PerkinElmer) or SuperSignal West Femto ECL kit (Thermo Scientific). Images were collected on the ChemiDoc Touch imagining system and analyzed using ImageLab software (Bio-Rad).