Determination of cAMP

Primary culture neurons were seeded into 96 well culture plates for 7 days. LV-shGLP-1R (10⁵ TU/ml) was added 4 days and exendin (9–39) (1 μM) was added 30 min prior to the addition of DMB (10⁻⁷–10⁻⁴ μM) or exendin-4 (10⁻¹⁰–10^−6.5 μM) to the culture wells. Cells were allowed to incubate for 0–30 min following treatment with DMB or exendin-4. A cAMP assay kit was then used to determine cAMP levels according to the manufacturer’s instructions. All data were subsequently normalized to the response of 1 mM GLP-1.