Requirement of dynamin2 S-nitrosylation on C86 and C607 for E. coli entry into BECs. (A) Bacterial invasion promotes the dynamin2 S-nitrosylation on C86 and C607 residues. BECs were transfected with HA-tagged dynamin2 (wild-type, C86A, C607A, or C86/607A), incubated with type 1 fimbriated HcRed-expressing E. coli for 1 h, and subjected to dynamin2 S-nitrosylation analysis by the biotin-switch method. CysNO-treated cell extracts were used as a positive control. NT, not transfected; WT, wild-type dynamin2; and SNO, S-nitrosylated protein. (B) BECs were transiently transfected with cDNAs encoding GFP-dynamin2 (wild type, K44A, C86A, C607A, or C86/607A) fusion proteins and subjected to invasion assays with the HcRed-expressing E. coli. Cells were washed, fixed in formalin, and counterstained with DAPI to visualize nuclei. 100 GFP- or GFP-dynamin2–expressing cells from each group were randomly selected, and the number of HcRed-expressing E. coli was counted. Each point represents the percent change in intracellular bacteria in comparison with GFP alone–expressing BECs. n = 3; *, P < 0.05 by ANOVA. Data represent means ± SEM.