Alphascreen surefire assay

Hepatocytes from ad libitum-fed male Sprague Dawley rats (body weight 200 g) were isolated in situ by a two-step perfusion technique as described previously [30 (link)]. The following day, the primary hepatocytes were used for assessment of insulin receptor (IR) phosphorylation (p-Tyr1150/1151) or Akt phosphorylation (p-Ser473) upon stimulation with increasing concentrations of HI, the dual GCGRi–insulin compound or the NC for 15 min, using the AlphaScreen SureFire assay kit (Perkin-Elmer, Denmark).

C2C12 myoblasts were seeded at a density of 6 × 10⁴ cells/well in 96-well plates and cultured at 37 °C and in a 5% CO₂-containing environment overnight. The following day, the medium was replaced with HBSS, and the cells were cultured for 3–4 h. Subsequently, the cells were incubated with various concentrations of amino acids for 15 min. After removal of HBSS, the cells were lysed using CelLytic MT Cell Lysis Reagent (C3228; Sigma, St. Louis, MO, USA) for 10 min, and the extract was subjected to an AlphaScreen SureFire assay (TGR70S500; PerkinElmer, Waltham, MA, USA) to detect phospho-p70 S6K (Thr 389) following the instructions of the manufacturer. The values were corrected for protein concentrations determined separately using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). All data are presented relative to those of control-deprived amino acids.