Experimental system for TfnR clustering. (A) Adenoviral bicistronic construct containing TfnR-AP and BirA-ER for tetracycline-repressible expression of bTfnRs. The Lys residue (marked in blue) within the AP is biotinylated by BirA-ER. (B) Schematic of site-specific biotinylation of TfnR. TfnRs with C-terminal acceptor peptide (AP) are biotinylated (bTfnR) by a coexpressed and ER-localized biotin ligase BirA. (C) SDS-PAGE of mixed streptavidin (SA) and purified heterotetrameric SA (eluted from a Ni-NTA column). The open circles and circles with a cross inside denote the A (active) and D (inactive/dead) monomers, respectively. (D) LCa-EGFP–expressing BSC1 cells infected with adenovirus encoding the TfnR-AP construct show colocalization of bound streptavidin with LCa-EGFP–labeled CCPs. Insets: enlarged channel separation of region highlighted by the white square indicating colocalization of streptavidin and LCa-EGFP (arrows). (E) Uptake of AF568-A1 in bTfnR-expressing cells in the presence or absence of 50 µg/ml Tfn assayed by flow cytometry (n = 4, average ± SD).