UPEC invasion promotes the dynamin2 S-nitrosylation at C86 and C607 residues. (A) S-nitrosylation of HA-tagged wild-type dynamin2 was increased with escalating doses of CysNO, as measured by the biotin-switch method (top). Equal amounts of cell lysate were immunoblotted with anti-HA antibody to show the equal protein loading across all lanes (bottom). (B) S-nitrosylation of dynamin2 is localized to the C86 and C607 residues of dynamin2. BECs were transiently transfected with cDNAs encoding HA-tagged dynamin2 (wild-type [WT], C86A, C607A, or C86/607A) and subjected to S-nitrosylation by 5 µM CysNO as described in A. (C and D) UPEC promote the time-dependent S-nitrosylation of endogenous dynamin2. BECs were incubated with UTI89 (C) or UTI89ΔFimH (D) for the indicated times, and endogenous dynamin2 was subjected to S-nitrosylation analysis using the biotin-switch method. 1 µM CysNO-treated cell extracts was used as a positive control, and total dynamin2 protein is shown on the bottom. (E) UTI89 induces the eNOS-dependent S-nitrosylation of dynamin2 in mouse bladder. Strips of the bladder transitional epithelium layer were harvested from wild-type and isogenic eNOS^−/− mice and incubated with UTI89. After 1-h incubation, S-nitrosylation of endogenous dynamin2 was assessed by the biotin-switch method (top). Treatment with CysNO served as a positive control, and total dynamin2 was similar between groups (bottom). SNO, S-nitrosylated protein.