JCS03217 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. miniLDLR endocytosis is blocked by ARH/Dab2 depletion. Cell surface proteins in siRNA-treated cells were biotinylated and precipitated using streptavidin-agarose beads. Surface miniLDLR (biotinylated) (A, top) was compared with total cellular miniLDLR (A, bottom). Double knockdown of Dab2 and ARH resulted in an increase of miniLDLR at the plasma membrane. (B-F) miniLDLR endocytosis in siRNA-treated cells. HA-antibody was used to label miniLDLR, and receptors internalized after 10 minutes at 25°C was detected by immunofluorescence. Z-projection stacks (4 μm) are shown, confirming that ARH/Dab2 and CHC siRNA blocked miniLDLR endocytosis.

• Supplemental Figure 2 -

  Fig. S2. FACS profiles of antibody-stained surface miniLDLR. Representative FACS profiles for control (A-C) and ARH/Dab2-depleted cells (D-F). After gating on viable cells, the population was homogeneous for fluorescence intensity, as demonstrated by the single peak in histograms, and shifted as a population upon internalization of surface-labeled receptors.

• Supplemental Figure 3 -

  Fig. S3. Tagged chimeric LDLR accumulates in filopodia in the absence of Dab2. (A-E’) Surface miniLDLR was detected by immunofluorescence in non-permeabilized cells, followed by permeabilization and staining for actin. Z-projection stacks of 1.2 μm are shown for representative cells. Filopodia are visible by actin staining (blue, arrowheads) together with miniLDLR (green) in the merged image (A-E). MiniLDLR is not present in filopodia in control and ARH-depleted cells (A’,B’), but can be seen along the length of the filopodia in Dab2, Dab2/ARH, and CHC knockdown cells (C’-E’). Bar, 5 μm.

• Supplemental Figure 4 -

  Fig. S4. MyoVI depletion also inhibits miniLDLR clustering. (A,B) MyoVI and Dab2 (green) localize to filopodia in HeLa cells, as indicated by colocalization with actin (red). Bar, 5 μm. (C) Depletion of MyoVI by siRNA assessed by western blot. (E-H) Surface miniLDLR distribution was examined in control and MyoVI-depleted cells. MyoVI (green) was compared with miniLDLR (red) to ensure depletion was effective. Single 0.2 μm sections at the bottom surfaces of cells are shown. In control cells, surface distribution of the miniLDLR was punctate as described previously (E,F). Depletion of MyoVI caused miniLDLR to become more diffuse, with less receptor in CCPs (G,H). This result was statistically significant, although not as severe as when Dab2 was depleted (**P<0.001) (D). Bar, 15 μm.

• Supplemental Figure 5 -

  Fig. S5. Depleting Dab2 and ARH does not affect miniLRP1 trafficking, and only slightly affects miniMegalin trafficking, in HeLa cells. siRNA was added to cells stably expressing either the miniLRP1 or miniMegalin receptor. Cell surface proteins were biotinylated and precipitated using streptavidin-agarose beads. Surface (biotinylated) miniLRP1 (A, top) and miniMegalin (C, top) were compared with total cellular miniLRP1 (A, bottom) and miniMegalin (C, bottom). (B,D) The ratios of surface:total miniLRP1 and miniMegalin were determined by densitometry, and values compared with control cells. Depletion of CHC resulted in an increase in both mini-receptors at the cell surface. Single- or double-depletion of Dab2 and ARH did not affect miniLRP1 distribution. Single depletion of Dab2 and ARH did not alter miniMegalin distribution, but double depletion of these adaptor proteins caused a slight increase in the amount of miniMegalin at the cell surface, suggesting a minor role for these proteins in megalin trafficking in HeLa cells.