Fig. 4

Quality evaluation of HPLC based PDE5A1-inhibitor screening assay. A The precision of HPLC-detected cGMP, which was 0.75 µg/mL, equal to the substrate concentration in the assay (n = 6). The pictures from the bottom to the top showed the the same sample was injected one to six. B The HPLC-detected analytes stability under 10 nM sildenafil in the assay within 24 h (n = 6). The pictures from the bottom to the top showed the peaks at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h. C The reproducibility of the assay inhibited by 10 nM sildenafil (n = 6). The peaks showed 6 samples prepared in the same way. D The signal window of 8 independent experiments (n = 8). E Dose–response curves of sildenafil acting on PDE5A1- hydrolyzed cGMP production in the PDE5A1-inhibitor screening assay. IC₅₀ = 14.22 nM ± 1.3 nM (n = 3)