Precision plus protein marker

Forskolin, MK-2206 and PI-103 were from Tocris (Avon, UK). Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence reagents were purchased from GE Healthcare (Piscataway, NJ, USA). [γ³²P]-labelled ATP was from Perkin Elmer (MA, USA). Precision Plus protein markers and SsoFast EvaGreen Supermix were from Bio-Rad (Hertfordshire, UK). Insulin-like growth factor 1 (IGF-1) was from Cell Signaling Technology (Hertfordshire, UK). Protease inhibitor cocktail tablets were purchased from Roche (Lewes, UK). Anti-HA-agarose, anti-FLAG-agarose, collagenase (from clostridium histolyticum type IV, Cat no. C5138), triiodothyronine, Bt₂-cAMP and lactate were from Sigma-Aldrich (Poole, UK). Infinity glucose assay kit was from Thermo Scientific (Essex, UK). Insulin and glucagon were from Novo Nordisk (Sussex, UK). Cell culture media and reagents including Dulbecco’s modified eagle medium (DMEM) and M199 with glutamax were from Life Technologies (Paisley, UK). All other chemicals unless specified were obtained from Sigma-Aldrich.

Flash-frozen tissues were prepared for sodium dodecylsulphate/polyacrylamide gel electrophoresis/western blotting as previously described using radioimmunoprecipitation assay buffer (31 (link),85 (link)). Protein expression was detected using combinations of primary and secondary antibodies as summarized in Table 3. Bands were visualized using electrochemiluminescence detection and quantified using FIJI Image-J v1.53a. Protein molecular weights were estimated using a calibration curve constructed using streptavidin-tagged Precision Plus Protein Markers (Bio-Rad, Hemel Hempstead, UK). Streptavidin-tagged markers were processed separately, yet in parallel, with the rest of the blot to prevent oversaturation of the electrochemical signal obtained from proteins detected in cell samples. Protein molecular weights were expressed as kDa; we have previously shown that such estimates have an error of 9.3% (86 (link)). Representative whole western blots for all target proteins are shown in Supplementary Material, Figure S2. Protein levels were normalized using the housekeeping protein β-tubulin and expressed as normalized protein expression. Protein expression for each group was calculated and expressed as normalized tubulin ratio, with error bars quoted as ±SEM for results obtained using patient tissue and as ± SD for results obtained using in vitro cell models.

For SDS-PAGE, 14-µL samples mixed with 5.3 µL Laemmli buffer and 0.6 µL 2-mercaptoethanol were heated to 95 °C for 5 min. After cooling, 12-µL aliquots were transferred to the wells of TGX (Tris-Glycine eXtended) 4–20% gels (Bio-Rad Laboratories, Hercules, CA, USA) in an electrophoresis chamber filled with 1:10 diluted TGS buffer (Bio-Rad Laboratories, Hercules, CA, USA). Quantitative albumin standards of 500, 250, 125, 100, and 50 µg mL⁻¹ (Thermo Fisher Scientific, Dreieich, Germany) were run alongside. The gels were analyzed using the ChemiDoc MP imaging system and Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). To determine the molecular weights of each band, Precision Plus Protein markers (Bio-Rad Laboratories, Hercules, CA, USA) were run in the first and last lanes.

SDS-polyacrylamide gels (10%) were loaded with 20 μg protein/lane and separated for an initial 10 min at 100 V followed by 80 min at 150 V and stained with Coomassie blue silver stain (20% methanol, 10% (w/v) ammonium sulfate, 1.6% orthophosphoric acid, 0.15% (w/v) Coomassie G250). Other gels were loaded with 10 μg proteins per lane followed by Western blotting (see below). Either Broad Range or Precision Plus protein markers (Bio-Rad) were used as size standards.