Figure 2.

Mutation of tyrosine 819/828 in human PROM1 reduces its interaction with Arl13b. A–D, polarized WT cells (MDCK)or those expressing human PROM1.s1 or PROM1.s2 splice variants or the corresponding Y819/828F mutants were subjected either to immunoisolation (IS) using paramagnetic beads conjugated to mAb against PROM1 (AC133) or anti-fluorescein isothiocyanate (FITC) antibody as negative control (A and B) or processed for indirect immunofluorescence followed by CLSM (C and D). For immunoisolation, the input (PROM1, 1:10; Arl13b, 1:40) and immunoisolated materials were probed for PROM1 and Arl13b by immunoblotting (A). The arrowhead and asterisks indicate plasma membrane and endoplasmic reticulum–associated species of PROM1, respectively, whereas arrows show Arl13b. Molecular mass markers (kDa) are indicated (left). The ratio of Arl13b/PROM1 (plasma membrane species only) immunoreactivities was quantified (B). 3–6 independent experiments were performed. Data are presented as mean and S.D. (error bars). Individual values are shown. **, p < 0.01; ***, p < 0.001 (two-tailed unpaired Student's t test). For immunofluorescence, cells were triple-immunolabeled for PROM1 (green), Arl13b (red), and AcTub (white). Nuclei (nu) were counterstained with DAPI (blue). The three-dimensional view (x-z orientation) was built from 38–45 x-y sections throughout the ciliary compartment (C, left panels) or x-y section (right top panels) and a composite of 5–10 x-y sections (right bottom panels) taken at the axoneme level (Ax, green bar) or at the base (red bar) of the primary cilium (C), respectively. The top view of three-dimensional reconstructions highlights Arl13 at the base of the cilium (D, dashed red line). Cells expressing either WT (left) or mutant (right) PROM1 are displayed. Green arrow, punctate PROM1 staining at the ciliary membrane of WT PROM1⁺ cells. Asterisk, PROM1 associated with microvillar membrane. Scale bars, 1 μm.