A. Rin expression in catecholaminergic tissue and cell lines. 20μg PC12 and SK-N-MC lysate and 150μg rat striatal lysate were resolved by SDS-PAGE and immunoblotted with mouse anti-Rin antibodies. B. Co-immunoprecipitations. DAT was immunoprecipitated from equivalent amounts lysate from PC12 cells transfected with either vector (−) or DAT (+). Control immunoprecipitations were performed using anti-transferrin receptor (TfR) antibodies or with Protein A/G beads alone. Bead eluents (Bead) and 1/10th supernatant volumes (Sup) were resolved by SDS-PAGE and immunoblotted with an anti-Rin antibody. Representative blots are shown (n=3). C. Co-immunoprecipitations from rat striatal synaptosomes. Immunoprecipitations were performed with solubilized rat striatal synaptosomes using Protein A beads coated with rabbit anti-DAT Abs or rabbit IgG alone. Eluents were resolved by SDS-PAGE and immunoreactive bands were detected with the indicated antibodies. A representative blot is shown (n=2).

Immunofluorescence. PC12 cells were co-transfected with DAT and CFP-Rin and were fixed, stained as follows: A. Cells were stained with anti-DAT and anti-GFP antibodies and imaged as described in Materials and Methods. Boxed indicates an enlargement of the areas indicated with white boxes in the merged images. B. Cells were labeled with Alexa594-CTX and were subsequently stained with anti-DAT and anti-GFP antibodies and imaged as described in Materials and Methods. C. Enlargement of the boxed areas from images in Figure 2B. Arrows indicate DAT immunoreactive pixels that co-localize with neither Rin (blue) nor CTX (red). Arrowheads indicate foci of DAT/Rin co-localization that also co-localize with CTX. D. Average data. DAT/Rin co-localization in CTX-positive (+CTX) and CTX-negative (−CTX) cell regions was measured at 10 independent thresholds per channel, as described in Materials and Methods. Average data at the median threshold are expressed as %DAT/Rin co-localization ±S.E.M. *Significantly different from −CTX, paired t test, p<.0001, n=16.

HEK 293 cells were transiently transfected with cDNAs encoding CFP- or YFP-tagged proteins as indicated and epifluorescence microscopy was performed two days post-transfection. A. The first and second columns show images obtained with CFP and YFP filter sets, respectively; the third column displays a corrected and normalized FRET image (N_FRET) established with PIXFRET. A look-up table of the colour code used is presented in the last column. All images shown are representative of 3–7 experiments. In all images, background fluorescence was subtracted. B. Averaged data. Normalized FRET efficiencies (“N_FRET values”) are given for cells expressing the following constructs: C-Rin and membrane-bound YFP (membr-YFP; n=15), Y-DAT and membr-CFP (n=25), C-TfR and Y-Rin (n=20), C-Rin and Y-Rin (n=16), C-SERT and Y-Rin (n=27), C-rGAT1 and Y-Rin (n=30), C-DAT and Y-Rin (n=24), C-Rin and Y-DAT (n=20), C-DAT and Y-DAT (n=23) and C-TfR and Y-TfR (n=37). All values were analyzed by one-way ANOVA with Dunn’s multiple comparison test. *Significantly different from (YFP-DAT and membr-CFP), (CFP-Rin and YFP-Rin) and (Rin and membr-YFP), p<.001. **Significantly different from (CFP-TfR and YFP-Rin), (CFP-Rin and YFP-Rin) and (Rin and membr-YFP), p<.001. See Table I for statistical analysis of complete data set.

GST pulldown assays. PC12 homogenate was incubated with either GST or GST-DAT(587–617), 1 hour, 37°C. Complexes were isolated with glutathione agarose and Rin was detected by immunoblot as described in Materials and Methods. A. Rin is isolated with the DAT carboxy terminus, but not GST alone. B. Concentration dependence. GST-DAT(587–617) was incubated with increasing concentrations of PC12 homogenate, 1 hour, 37°C followed by isolation of complexes with glutathione agarose. Top: Representative immunoblot probed for Rin. Bottom: Averaged data. Results are expressed as % maximal Rin signal vs. log PC12 homogenate concentration (mg/ml) and were fit to a sigmoidal dose response curve (r²=.98, n=3).

A–C. Co-immunoprecipitations. PC12 cells stably expressing either wildtype DAT (A) or DAT587–590(4A) (B) were pre-treated with either 1μM BIM or vehicle, 20min, 37°C followed by treatment with either vehicle or 1μM PMA, 30min, 37°C. Cells were lysed and DAT was immunoprecipitated from equivalent amounts of cellular protein. Immunoprecipitants were resolved by SDS-PAGE and immunoblotted for both DAT and Rin. (Left) Representative immunoblots. (Right) Averaged data. Rin signals from non-saturating bands were normalized to DAT signals and are expressed as %vehicle signal ±S.E.M. *Significantly different from vehicle control (p<0.002, One-way ANOVA with Tukey’s multiple comparison test, n=4). C. Basal Rin interaction with wildtype vs. 587–590(4A) DAT. Left: Representative immunoblot. Right: Averaged data. Rin signals from non-saturating bands were normalized to DAT signals and expressed as % wildtype signal ±S.E.M. *Significantly different from wild type DAT (*p<0.0005, Student’s t test, n=3). D. GST pulldowns. GST fused to either wildtype or DAT 587–590(4A) carboxy termini were induced and isolated on glutathione Sepharose as described in Materials and Methods. Equivalent amounts of GST-DAT fusion proteins were incubated with PC12 homogenates and bound proteins were resolved by SDS-PAGE and immunoblotted for both Rin and GST. Left: Representative immunoblot. Right: Averaged data. Rin densities were normalized to total GST pulled down for each sample and are expressed as %Rin ±S.E.M isolated as compared to wildtype GST-DAT. *Significantly different from wildtype GST-DAT (p<0.04, Student’s t test, n=5).

Internalization assays. PC12 cells were co-transfected with DAT and the indicated Rin or control cDNAs, and were assayed 48–72 hours post-transfection. Relative DAT internalization rates over 10 minutes were measured by reversible biotinylation during treatment ±1μM PMA, 37°C as described in Materials and Methods. Top: Representative immunoblots showing total surface DAT at time=0 (T), strip controls (S), and internalized DAT under vehicle- (V) and PMA-treated (P) conditions. Bottom: Average Data. Data are expressed as %DAT internalized/10 min as compared to total surface DAT at t=0, ±S.E.M. *Significantly different from vehicle-treated control, Student’s t test, n=4 (*p<.001, **p<.05).

Immunofluorescence. PC12 cells were co-transfected with DAT and the indicated Rin or control cDNAs, and were assayed 48 hours post-transfection. Cells were treated ±1μM PMA, 30′, 37°C and were fixed, stained with anti-DAT or anti-GFP (to detect CFP-Rin) antibodies, and imaged as described in Materials and Methods. Planes through the cell center are shown and are representative of 12–16 cells imaged/condition in instances where PMA-induced internalization was apparent (n=4).

hRin knockdown and antibody internalization assay. A. hRin knockdown. hRin-directed shRNAs were screened in HEK cells, 72 hours post transfection. Top: Representative immunoblot showing Rin levels in vector-, scrambled shRNA, and hRin228-transfected cells, with actin probed as a loading control. Bottom: Averaged data. Data are expressed as %Rin levels as compared to vector-transfected cells (normalized to actin loading control). *Significantly different from vector control, one-way ANOVA with Dunnett’s posthoc analysis, p<.05, n=5. B, C. Antibody internalization assay. SK-N-MC cells were co-transfected with EL2-HA-DAT and the indicated constructs were assayed 72 hours post transfection. Cells were labeled with HA antibody and DAT internalization was assessed for 15 min, 37°C in the presence of 1μM PMA as described in Materials and Methods. Cells expressing shRNA constructs were identified by GFP co-expression. Optical z-stacks were collected and deconvolved as described in Materials and Methods. B. Averaged data. Data are expressed as % cells exhibiting PMA-induced intracellular DAT puncta ±S.E.M. for each of the indicated transfection conditions. *Significantly different from vector-transfected control, p<.05, One-way ANOVA with Dunnett’s multiple comparison test, n=3. C. Representative images. Planes through the cell center are shown for two cells per condition, and are representative of 30–34 cells imaged/condition over 3 independent experiments.

SK-N-MC cells were co-transfected with DAT and the indicated constructs and [³H]DA uptake was assessed 72 hours post transfection. Cells were treated ±1μM PMA, 30′, 37°C, followed by addition of [³H]DA as described in Materials and Methods. Averaged data are shown and are expressed as % uptake of vehicle treated cells ±S.E.M. *Significantly different from vehicle-treated control, (p<0.03, Student’s t test, n=4).