Insulin Receptor-A Phosphorylation Assay

Insulin receptor-A phosphorylation was detected essentially as described by Ref. (6 (link)). Briefly, R^–IR-A cells (5 × 10⁴ cells/well) were plated in a 96-well flat-bottom plate and grown overnight at 37°C, 5% CO₂. Cells were starved in serum-free medium (SFM) for 4 h before treatment with insulin, IGF-II, qIGF-I, or S597 in 100 μl of Dulbecco’s minimal essential medium with 1% bovine serum albumin for 10 min or in a time course (0, 2, 5, 8, 12, 20, 30 min) at 37°C, 5% CO₂. Cells were lysed with ice-cold lysis buffer containing 2 mM Na₃VO₄ and 100 mM NaF, and receptors were captured onto white Greiner Lumitrac 600 96-well plates pre-coated with anti-IR antibody 83-7 (500 ng/well) (34 (link)) and blocked with 20 mM Tris, 150 mM NaCl, and 0.1% (v/v) Tween 20 (TBST)/0.5% bovine serum albumin. Following overnight incubation at 4°C, the plates were washed three times with TBST. Phosphorylated receptor was detected by incubation with EU-pY20 (76 ng/well) at room temperature for 2 h. Wells were washed four times with TBST, and time-resolved fluorescence was detected as described above. Assays were performed in triplicate at least three times.