Figure 7.

Effect of endothelial cell BH4 deficiency on the ratio of eNOS dimers to monomers and eNOS glutathionylation. (A) Representative Western blot for GTPCH, eNOS protein in sEND.1 mouse endothelial cell line treated with non‐specific Gch1 siRNA (NS) or specific Gch1 siRNA (Gch1 siRNA). (B) Intracellular BH4, BH2 and total biopterins, measured by HPLC, were significantly reduced in Gch1 specific siRNA cells compared with non‐specific siRNA cells. (C) Ratio of BH4 relative to oxidized biopterin species (BH4:BH2 + B). (D) Quantification data, measured as band density of eNOS to β‐tubulin. (E) Representative Western blot for eNOS dimer/monomer in sEND.1 mouse endothelial cell line treated with non‐specific Gch1 siRNA (NS) or specific Gch1 siRNA (Gch1 siRNA) using a low‐temperature gel. (F) Quantification data, measured as band density of dimer to monomer protein in NS and Gch1 siRNA. Western blot analyses are representative of three separate experiments. (G, H) Representative Western blot for eNOS‐glutathionylation and eNOS in sEND.1 with non‐specific and specific siRNA knockdown with or without supplementation of 10 μmol·L⁻¹ sepaipterin by immunoprecipitation. Non‐specific sEND.1 knockdown with dithiothreitol (DTT; 1 mmol·L⁻¹) was used as negative control for eNOS‐glutathionylation. Western blot analyses are representative of six separate experiments. Data are expressed as mean ± SEM; n = 6. *P < 0.05, significantly different from non‐specific siRNA).