Hook1 colocalizes with CD44, CD98, and CD147 on tubular endosomes. (A) HeLa cells were incubated with anti-CD147, anti-CD44, or anti-CD98 for 30 min at 37°C to allow the internalization of the cargo-bound antibodies. After internalization, cells were treated for 1 min with 10 µg/ml digitonin and then fixed. Endogenous Hook1 was localized using a rabbit anti-Hook1 antibody. Arrows point to tubular endosomes. (B) HeLa cells overexpressing HA-Hook1 were incubated with anti-CD147 antibody for 30 min at 37°C and processed for immunofluorescence. Enlargements of the two boxed regions are shown below. Arrows in the middle row indicate tubes, and arrows in the bottom row indicate swollen regions of the tubes. (C) After antibody internalization, endogenous CD147 (green; Alexa Fluor 488) and endogenous Hook1 (red; Alexa Fluor 568) were visualized using super-resolution fluorescence microscopy (SIM). This image is one projection of a 3D SIM image of a region of interest in the cell where CD147 colocalizes with Hook1 on tubular endosomal structures. Insets show enlarged views of the boxed regions. (D) Projection of a 3D image (from C, box 1) from 0° to 18° with 2° increments showing colocalization between CD147 (green) and Hook1 (red) at the end of a tubular endosome. (E) Montage of a 3D image (from C, box 2) from 0° to 10° with 2° increments showing CD147 (green) and Hook1 (red) together on an endosome (F). Endogenous Hook1 (red) and internalized CD98 (green) colocalized in tubular endosomes. Shown is a montage of a projection of a 3D image from 0° to 12° with 2° increments. Arrows in C–F point to Hook1 and cargo colocalization positions. Bars: (A–C) 10 µm; (A, insets) 3 µm; (B, insets) 5 µm; (C, inset) 3 µm; (D) 2 µm; (E and F) 1 µm.