Cgmp elisa kit

L-[¹⁴C]arginine and L-[¹⁴C]citrulline were obtained from PerkinElmer Inc. (Santa Clara, CA, USA). Antibodies for phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, IRS-1, phospho-IRS-1 (Ser³¹²), phospho-IRS-1 (Ser⁶¹⁶), phospho-IRS-1 (Ser⁶¹²), phospho-IRS-1 (Tyr⁶³²), phospho-IRS-1 (Tyr⁶²⁸), the p85 subunit of PI3K, phospho-protein kinase B (AKT; Ser⁴⁷³), AKT, eNOS, phospho-eNOS(Ser¹¹⁷⁷), and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PD98059 (a reversible MEK1 inhibitor), SP600125 (inhibitor of JNK), N^G-nitro-L-arginine (L-NNA, inhibitor of NOS), cGMP ELISA kit and 20-Hydroxyeicosatetraenoic acid (20-HETE) were obtained from the Cayman Chemical Company (Ann Arbor, MI, USA). Other reagents were obtained from Sigma (St. Louis, MO, USA).

Four-week-old mice were treated with vehicle, DEX, or CNP-53, or CNP-53 and DEX for 3 days. These mice were sacrificed 30 minutes after the last injection and their lumber vertebrae were resected. Samples were homogenized in 5% trichloroacetic acid (TCA) and TCA was extracted into water-saturated ether. The obtained cGMP solutions were acetylated to increase the sensitivity of the following assay. cGMP levels of the lumbar vertebrae were measured by cGMP ELISA kit (No. 581021, Cayman Chemical, Michigan, USA).
For in vitro studies, ATDC5 cells were plated at 1.0 × 10⁵ cells/well in 6-well tissue culture plates and cultured with 10 µg/ml bovine insulin (No. 10516, SIGMA) for 14 days to differentiate into proliferative chondrocytes which are abundant in NPR-B^{50 (link)}. After differentiation, ATDC5 cells were incubated with vehicle or 10⁻⁷ M DEX for 30 minutes, and then vehicle or 10⁻⁷ M CNP-22 (No. 4229-v, PEPTIDE INSTITUTE) were added into the medium for 24 hours. Culture medium was acetylated to increase the sensitivity of the following assay and cGMP levels were measured directly by cGMP ELISA kit.

Protein lysates were obtained as described above. The cGMP ELISA kit was purchased from Cayman Chemical. A total of 30 μg of protein was diluted to 100 μL in incubation buffer consisting of 50 mM Tris-HCl (pH 7.5, Fisher Scientific), 4 mM MgCl₂ (Fisher Scientific), 0.5 mM 3-isobutyl-1-methylxanthine (Enzo), 7.5 mM creatine phosphate (Sigma-Aldrich), 0.2 mg/mL creatine phosphokinase (Sigma-Aldrich), 10 mM sodium nitroprusside (Sigma-Aldrich), and 1 mM GTP (Sigma-Aldrich) for 10 min at 37 °C. The reaction was terminated with 900 μL of ice-cold 0.1 N HCl (Sigma-Aldrich) and then placed in a 90 °C water bath for 3 min to denature the protein. Samples were centrifuged for 15 min at 2000× g to precipitate protein. The supernatant was collected, dried in a speed vacuum, and resuspended in cGMP EIA buffer (Cayman Chemical, Ann Arbor, MI, USA). Samples were diluted 1:2 in EIA buffer for optimal calculation. Absorbance was detected at 405 nm using an iMark automated plate reader (BioRad, Hercules, CA, USA).

The purified haem-free sGC were diluted with 20 mM triethanolamine (TEA pH7.6), 300 mM NaCl and subjected to the enzyme activity assay. The reaction system contained 14 nM sGC, 60 mM TEA (pH 7.6), 150 mM NaCl, 0.5 mM DTT, 5 mM MgCl₂, 200 µM GTP and various chemical drugs as indicated in a final volume of 20 μl. The final concentration of DMSO was 1% (v/v). The assay mixture was incubated at 25 °C for 10 min, and then 80 μl 125 mM Zn(OAc)₂ and 100 μl 125 mM Na₂CO₃ were added to stop the reaction. The GTP-ZnCO₃ precipitation was removed by centrifuged at 17,000 g for 5 min, and the supernatants were used for the quantification of cGMP with the cGMP ELISA Kit (Cayman Chemical) according to the instructions. For activity assay in Figs. 3a, b and 4j, 0.5 mM DTT in the assay mixture was omitted and the reaction temperature was changed to 37 °C. Each assay was repeated for at least three independent reactions.

cGMP levels were measured in homogenates from goldfish hearts perfused under both normoxic and hypoxic conditions. Samples were treated with 5% trichloroacetic acid on ice and centrifuged at 1500× g for 10 min. The supernatant was extracted three times with 5 volumes of diethyl ether saturated with water; the aqueous phase was collected and used for cGMP measurements, using a commercial enzyme immunoassay kit (cGMP ELISA Kit; Cayman Chemical, Ann Arbor, MI, USA).

ATDC5 cells were plated at 1.0 × 10⁵ cells/well in 6-well tissue culture plates. ATDC5 cells were differentiated into proliferative chondrocytes by incubation with 10 μg/ml bovine insulin (No. 10516, SIGMA) for 14 days. Differentiated ATDC5 cells were incubated with vehicle or 10⁻⁷ M CNP-22 for 30 minutes. Culture media were acetylated and their cGMP level were measured using the cGMP ELISA kit (No. 581021, Cayman Chemical, Michigan, USA).