Fig. 3
STEAP3 interacts with the viral receptor during viral entry. (A) HEK293T cells were transfected with pIRES2-Flag-STEAP3 and pcDNA3.1-His/Myc-SCARB2 for 48 h. The total cell lysate was collected, and an IP assay was performed using anti-Flag antibody and separated by SDS-PAGE. The interaction of His/Myc-SCARB2 was analyzed using anti-His antibody. (B) The total cell lysate was collected from RD cells infected with EV-A71 (MOI = 10−3) for various intervals and subjected to IP assay with anti-STEAP3 or anti-IgG antibodies. The immunoprecipitates were then separated and analyzed with anti-SCARB2 antibody. The interaction between STEAP3 and SCARB2 was quantified by the ratio of SCARB2 expression in the precipitates to the expression of SCARB2 in total cell lysates (n = 3). *p < 0.05 compared to RD cells without EV-A71 infection. Results are presented as the mean percentage (± s.d.). (C) ACE2-HEK293T cells were transfected with pIRES2 empty vector or pIRES2-Flag-STEAP3 for 48 h. The total cell lysate was collected and subjected to an IP using anti-Flag antibody. The binding of ACE2 and STEAP3 was further analyzed in the precipitates by western blot using anti-ACE2 antibody. (D) The total cell protein was extracted from Caco-2 cells and immunoprecipitated with anti-STEAP3 or control IgG antibodies. The interactions of ACE2 in anti-STEAP3 precipitates were further analyzed using anti-ACE2 antibody. (E) Colocalization between ACE2 and STEAP3 in Caco-2 cells was detected by double immunofluorescence staining with anti-ACE2 (red) and anti-STEAP3 (green) antibodies and counterstained with DAPI (blue). Images were visualized using an Andor Dragonfly confocal microscope. Scale bar = 10 μm. The highlighted area in the inset represents a 2.5-fold magnification of the region indicated by the arrowhead. Scale bar = 1.6 μm. The uncropped and unprocessed blots are presented in Supplementary Fig. S17.
