Impaired TF protein expression in endothelial cells transduced with adenovirus carrying TF mutant, TF_C186S, TF_C209S, or TF_C186S/C209S. HUVECs were transduced with control adenovirus (β-Gal), adenovirus encoding wild-type TF, TF_C186S, TF_C209S, or TF_C186S/C209S (25 MOI/cell), and TF expression levels were analyzed by various methods: confocal microscopy (A), fluorescence-activated cell sorting analysis (B), immunoblot analysis (C), in situ ELISA to measure cell-surface TF (D), and ELISA to measure total TF antigen levels (E). (A) HUVECs, nonpermeabilized (top) or permeabilized with 0.05% Triton X-100 for 10 minutes (bottom) were immunostained with polyclonal anti-TF IgG (10 μg/mL) followed by Oregon Green–conjugated secondary antibodies. Immunofluorescence was analyzed by confocal microscopy at 2 different gain settings (high gain, 840; low gain, 630) to capture differences in TF expression in cells expressing wild-type TF or TF mutant. (B) Intact, nonpermeablized HUVECs were stained with anti-TF polyclonal antibodies, followed by FITC-conjugated secondary antibodies, and the cells were analyzed for the fluorescence by flow cytometry. Wild-type TF, pink; TF_C186S, cyan; TF_C209S, red; TF_C186S/C209S, orange; β-gal, green. (C) Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, either nonreducing (left) or reducing (right) conditions, and immunoblotted for TF with the use of polyclonal anti-TF antibodies. (D) TF expression levels at the cell surface were measured by in situ ELISA with the use of polyclonal anti-TF IgG or TF mAb, TF96B4 (n = 4; mean ± SEM). (E) Total TF antigen levels in cell lysates were measured by ELISA (n = 3, mean ± SEM).