ARH localization in TfnR-positive REs depends on AP-1B. LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA cells were grown on coverslips and infected with defective adenoviruses encoding ARH-GFP. After 24 h, cells were fixed and stained with anti-HA (A) or anti-TfnR (B) antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. Insets show 2× magnifications of the boxed regions. A′ and B′ show representative fluorescence intensity profiles through a region in the boxed area where noncoincidental peaks are marked by arrowheads (ARH-GFP) or arrows (AP-1A or TfnR). Bars, 10 µm. (C) For quantitation, confocal raw data were analyzed using Volocity software to determine the degree of colocalization between ARH-GFP and AP-1A (n = 64) or AP-1B (n = 71) as well as TfnR in LLC-PK1::µ1A-HA (TfnR[A], n = 54) or LLC-PK1::µ1B-HA cells (TfnR[B], n = 60). Data represent mean values from at least three independent experiments, error bars indicate SD. *, P < 0.0001. (D) Filter-grown MDCK cells were infected with defective adenoviruses encoding ARH-GFP. 24 h later, cells were stained for TfnR. Specimens were analyzed by confocal microscopy and representative xy, xz, and yz images are shown. Bar, 5 µm.