Fig. 1. Northern blot analysis of SNX17 expression in murine tissues. (A) Total RNAs (30 µg) from various tissues from male and female Balb/C mice were separated by electrophoresis on a 1.5% agarose gel and blotted as described in Materials and methods. Hybridization was performed using the full-length SNX17 probe. (B) Methylene Blue staining was used as the loading control.

Fig. 2. Interaction of SNX17 with members of the LDLR family. (A) Extracts of 293 cells expressing HA-tagged SNX17 (lane 1) or transfected with empty plasmid (lane 2) were loaded directly onto the gel, or incubated with various GST–receptor tail fusion proteins (lane 3, ApoER2 tail without the proline-rich insert; lane 4, proline-rich insert; lane 5, ApoER2 tail with the proline-rich insert; lane 6, VLDLR tail; lane 7, LDLR tail; lane 8, LRP tail; lane 9, megalin tail) pre-bound to glutathione–Sepharose for 2 h at 4°C. Bound proteins were eluted from the beads and analyzed by electrophoresis and western blotting with the anti-HA antibody. (B) Extracts of 293 cells stably expressing myc-tagged SNX17 and transiently transfected with an expression plasmid for ApoER2Δ4–6 (Brandes et al., 2001) were loaded onto the gel (lanes 1 and 2). Lane 3: an aliquot of the extract was incubated with anti-ApoER2. Immune complexes were precipitated using protein A–Sepharose, eluted from the beads and loaded onto the gel. The presence of myc-tagged SNX17 and ApoER2 was analyzed by western blotting using the indicated antibodies. (C) Extracts of MEFs grown in LPDS for 24 h and transfected with an expression plasmid for myc-tagged SNX17 (MEF-SNX17) (lanes 1 and 3) or transfected with the empty plasmid (mock; lane 2) were loaded directly onto the gel, or incubated with anti-myc antibody (9E10). Immune complexes were precipitated using protein G–Sepharose, eluted from the beads and loaded onto the gel (lane 4, MEF-SNX17; lane 5, mock). The presence of myc-SNX17 and LDLR was tested by western blotting using the indicated antibodies.

Fig. 3. Effect of SNX17 on LDLR-mediated binding, uptake and degradation of LDL. MEFs were stably transfected with SNX17 expression plasmid or with the empty plasmid and cultivated in LPDS 24 h prior to the experiments. The monolayers were incubated with the indicated concentrations of [¹²⁵I]LDL (specific activity 200 c.p.m./ng) in the absence (total binding) or presence (non-specific binding) of 500 µg/ml unlabeled LDL for 5 h at 37°C. Binding, uptake and degradation of [¹²⁵I]LDL were determined as described in Materials and methods. High affinity values were calculated by subtraction of values for non-specific binding from those for total binding. Each data point represents the average of duplicate incubations.

Fig. 4. Analysis of the localization of SNX17 by isopycnic centrifugation on a continuous density gradient. MEFs expressing myc-tagged SNX17 were cultured in LPDS for 24 h prior to the experiment. Cells were harvested, and a post-nuclear supernatant prepared and loaded on top of a 10–40% continuous sucrose gradient as described in Materials and methods. After centrifugation, fractions were collected from the bottom of the tube and 30 µl of each fraction were subjected to electrophoresis. The respective proteins were localized by western blotting using anti-myc antibody for SNX17, and the corresponding antibodies against the respective proteins described in Materials and methods. Fractions are numbered according to increasing densities.

Fig. 5. Immunofluorescence analysis of SNX17 in MEFs and localization with reference to EEA1, rab4, rab5 and rab7. MEFs stably transfected with an expression plasmid carrying the cDNA for 9xmyc-tagged SNX17 were grown on culture slides (Becton Dickinson) and stained for SNX17 (green) and the indicated marker proteins (EEA1, rab4, rab5 and rab7; red) as described in Materials and methods. Primary antibodies were visualized with the secondary antibodies Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG. Bar: 10 µm.

Fig. 6. Immunofluorescence analysis of LDL–rhodamine uptake by SNX17-expressing MEFs. MEFs stably transfected with an expression plasmid carrying the cDNA for 9xmyc-tagged SNX17 were grown in medium containing LPDS on culture slides (Becton Dickinson) and incubated with LDL–rhodamine at 4°C. Cells were then either fixed immediately (0 min) or the medium was changed and the slides were transferred to 37°C and fixed after the indicated time intervals. Cells were stained for SNX17 (green) as described in Materials and methods. Bar: 10 µm.

Fig. 7. Binding of SNX17 and Dab1 to the intracellular domain of ApoER2. Microtiter plates (96-well) coated with a GST fusion protein containing the intracellular domain of ApoER2 were incubated (A) with the indicated amounts of purified Dab1–MBP expressed in E.coli or (B) with cell extracts derived from 293T cells expressing myc-tagged SNX17. Bound Dab1–MBP was visualized with the combination of an anti-MBP antibody and HRP-conjugated goat anti-rabbit IgG. Bound myc-SNX17 was visualized using the combination of an anti-myc antibody and HRP-conjugated goat anti-mouse IgG as described in Materials and methods. The color reaction was monitored by measuring absorption at 450 nm. (C) Competition of SNX17 binding by Dab1 was measured at two selected concentrations of myc-SNX17 (corresponding to 25 and 40% of cell lysate present in the incubation medium, respectively) in the absence and presence of 2 µg/ml Dab–MBP.

Fig. 8. Interaction of SNX17 and Dab1 with ApoER2. (A) Extracts of 293 cells expressing ApoER2, 9xmyc-SNX17 and Dab1-HA were subjected to SDS–PAGE under reducing conditions and transferred to a nitrocellulose membrane. The presence of the expressed proteins was visualized by western blotting using anti-ApoER2 (lane 1), anti-myc (9E10; lane 2) and anti-HA (lane 3) antibody, respectively. Binding of the primary antibodies was visualized with HRP–goat anti-rabbit (lane 1) and HRP–goat anti-mouse (lanes 2 and 3) as described in Materials and methods. (B) Extracts of 293 cells expressing ApoER2, 9xmyc-SNX17 and Dab1-HA (lane 4) or mock-transfected cells (lane 5) were incubated with anti-myc–Sepharose. Immune complexes were eluted from the beads, and loaded onto the gel. The presence of myc-tagged SNX17 was visualized by western blotting using anti-myc antibody. (C) Extracts of 293 cells expressing ApoER2, 9xmyc-SNX17 and Dab1-HA (lane 6), mock-transfected cells (lane 7), cells expressing Dab1-HA (lane 8), cells expressing Dab1-HA and ApoER2 (lane 9) or cells expressing Dab1-HA and 9xmyc-SNX17 (lane 10) were incubated with anti-myc–Sepharose. Immune complexes were eluted from the beads, and loaded onto the gel. The presence of Dab1-HA was visualized by western blotting using anti-HA antibody and HRP-conjugated goat anti-mouse IgG as described in Materials and methods.