cGMP assay

The cGMP Direct Immunoassay Kit provided by R&D Systems was used according to the manufacturer’s instructions to quantitatively determine the cGMP expression.²¹ The cell lines were cultured for 24 hours to reach almost 80% confluence; then, they were treated with HAWE in a time-dependent manner (0, 4, 8, 12, and 24 hours). Afterward, the cells were lysed using a cell lysis buffer and were transferred to a 96-well plate coated with a goat anti-rabbit antibody. The excess conjugate and the unbound sample were removed via washing, and then the substrate solution was added to each well and incubated for 30 minutes at room temperature. Then, stop solution was added to each well. Finally, a microplate reader (Stat Fax 2100; Awareness Technology, Los Angeles, CA, USA) was used to measure cGMP at a 450-nm wavelength.