Camp glo assay kit

The intracellular cAMP concentration was measured using the cAMP-Glo assay kit from Promega according to the manufacture's instruction. The cAMP standard curve was generated using purified cAMP, from which the relative intracellular level of cAMP was inferred. For each drug treatment, 3 biological repeats were used, and each experiment was repeated 2–3 times.

Cyclic adenosine monophosphate (cAMP) accumulation in cells was determined using cAMP-Glo assay kit (Promega, Madison, WI, USA). In brief, R2C cells were plated in F12 medium on 96-well dishes at a density of 1 × 10⁴ for 24 h. The cells were disposed with 2 mmol/L 1,3-DCP with or without various concentrations of C3G (5, 10, 20, and 40 μmol/L) for 4 h. After 20 min of treatment with induction buffer [PBS containing the phosphodiesterase inhibitors IBMX (0.5 mM) and Ro 20-1724 (0.1 mM); Sigma–Aldrich, St. Louis, MO, USA], the reaction was stopped by cAMP-Glo Lysis Buffer. Then, the kinase reaction was performed using 40 μL cAMP Detection Solution for 20 min. At the end of the kinase reaction, 80 μL Kinase-Glo Reagent was added and incubated for 10 min, then a plate-reading luminometer (Biotek, Synergy HT, Winooski, VT, USA) was used for measurement.

To assess intracellular cAMP concentration, cells were cultured in 96-well plates at a density of 1 × 10⁴/well. The cAMP level was quantified using the cAMP-Glo Assay Kit (Promega, Cat# V1501) following the manufacturer’s instructions.

The assays utilize the cAMP-Glo Assay kit purchased from Promega (Fitchburg, WI, cat. no. V1501). For cAMP measurement in cells, chemicals were added 24 hr before measurement. On the day of measurement, change medium containing compound into PBS with phosphodiesterase inhibitors, and incubate 2 hr and then the procedures were performed following manufacture's instruction. Purified cAMP provided by the kit was diluted into different concentrations to plot the standard curve. The slope of the standard curve was utilized to determine the cAMP concentration change per unit change of the signal. For PKA activity measurement, compounds diluted in Opti-MEM at indicated concentrations were directly added into 384 well plates, and then the experiments were performed following manufacture's instruction.

The intracellular cAMP concentration was measured using the cAMP-Glo assay kit (Promega, V1501) according to the manufacturer’s instruction. The cAMP standard curve was generated using purified cAMP, from which the relative intracellular level of cAMP was inferred. For each drug treatment, three biological repeats were used, and each experiment was repeated 2–3 times.

The cAMP levels in cells and the xenograft tumor tissues were quantified according to the manufacturer’s instructions using a cAMP-Glo™ assay kit (Promega, USA). The enzyme activities of PDE4D isoforms that were immunoprecipitated from cells and xenograft tumor tissues were quantified according to the manufacturer’s instructions using a PDE-Glo™ phosphodiesterase assay kit (Promega).