Fig. 2.

HIV-1 envelope binds TIM-4 protein.

(A) 1 × 10⁶ HEK-293 cells were either untransfected or transfected with either psh-CMV or psh-CMV-HIV-Env (2.5 μg). The cell lysates were collected and protein concentration was determined. TIM-4-His₆ protein was coated on ELISA plates at 150 ng/well. The plate was washed and blocked, followed by the application of three different conditioned cell lysates (60 μg/well, n = 3). HIV-1-specific human monoclonal antibody 2F5 or 4E10 was applied to the plate after washing and blocking. Goat anti-human IgG-HRP was applied to the plate, which was then treated with o-phenylenediamine dihydrochloride peroxidase substrate. Signals were read at OD450 nm and expressed as fold change. (B) ELISA plates were incubated with 200 ng/well of recombinant gp140 proteins (UG37/HIV/Clade A, SF162/HIV/Clade B, CN54/HIV/Clade C, UG21 HIV/Clade D, or BR29/HIV/Clade F). The plates were washed and blocked, followed by the application of TIM-4-His₆ protein (400 ng/well, n = 3) or binding buffer (control). Next, mouse anti-His₆ monoclonal antibody was applied to the plate after washing and blocking. Goat anti-mouse antibody IgG-HRP was applied to the plate, which was then treated with OPD peroxidase substrate and read at OD450 nm. Background OD values were ~ 0.004. (**) = P ≤ 0.01, (***) = P ≤ 0.001, (****) = P ≤ 0.0001.