(A) NAC treatment (10 mg/ml; 3 weeks) increased whole-lung HIF 1–DNA binding activity. Complexes were supershifted (ss) with anti–HIF 1β and eliminated with excess cold probe (P). (B) SNOAC (1 μM), like GSNO (5, 13, 38), increased normoxic HIF 1α expression in nuclear extracts isolated from primary murine pulmonary endothelial cells. NAC alone (50 μM) did not affect HIF 1α expression. β-Actin was used as a protein load control. (C) In BPAECs transfected with HA-tagged HIF 1α and dominant-negative His-6-Myc–tagged ubiquitin (DN-Ub), ubiquitinated proteins were isolated using a nickel column and immunoblotted for HIF 1α. Both hypoxia and GSNO (G; 10 μM) inhibited HIF 1α ubiquitination relative to normoxia. (D) In COS cells cotransfected with HA-tagged HIF 1α and FLAG-tagged pVHL, GSNO (10 μM) prevented the coimmunoprecipitation of HIF 1α with pVHL. (E) S-nitrosylation of pVHL by SNOAC (5 μM) in equal protein aliquots isolated from HeLa cells was identified by biotin substitution (49); in the absence of ascorbate, S-nitrosylated pVHL was not detected. (F) Similarly, SNOAC and GSNO (5 μM) increased pVHL S-nitrosylation in pVHL-overexpressing 786-O cells. (G) C162, but not C77, was identified by biotin substitution to be S-nitrosylated in BPAECs transfected with wild-type cysteine 77 to serine mutant (C77S), C162S, or combined C77S/C162S pVHL exposed to SNOAC (1 μM). Native pVHL and MAPK immunoblots represented the pVHL expression and protein load controls, respectively. All in vitro treatments were for 4 hours.