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MBC Video for Molecular Biology of the Cell: Volume 0, Issue 2003 -- E02-11-0767

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Supplemental Material

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Supplementary Figure Legends: Aschenbrenner et al.

Figure 1. Immunoblot analysis using myo6-specific antibodies. Extracts of ARPE-19 cells were separated by SDS-PAGE and probed with rabbit affinity purified anti-M6tail antibody (Hasson & Mooseker, 1994), rabbit affinity purified anti-myo6motor antibody (this manuscript) and Mouse affinity purified anti-M6tail antibody (Hasson et al., 1997). A single 145kDa band (arrow) is detected by all three antibodies. Molecular weight markers, in kilodaltons, are shown to the left.

Figure 2. As fluid phase uptake follows an endocytic pathway similar to that shown for transferrin and we hypothesized that dextran uptake could also be blocked after overexpression of the tail domain of myo6. ARPE-19 cells were transfected with GFP (a,b), GFP-M6full (c,d) and GFP-M6tail (e,f) and incubated for various times with rhodamine-conjugated dextran before fixation. An example after 10 mins of rhodamine-dextran uptake is shown here. Dextran fluorescence is shown in panels a,c and e and GFP fluorescence is shown in panels b,d and f.

Both the abundance and the size of dextran-labeled compartments were similar between transfected and untransfected controls cells. These results suggested that the block in uptake of R-Tsfn was not due to a general defect in cell uptake, and that perhaps the block in R-Tsfn uptake was due to a specific requirement for myo6 in the transport of the Tsfn-R through the endocytic pathway. Scale bars = 10mu m.

Figure 3. GIPC is not a component of clathrin-coated pits in ARPE-19 cells. Cells were double-labeled for immunofluorescence staining with antibodies directed to the GIPC and to the clathrin adaptor protein AP-2. (a,b) Low magnification view showing an example of the whole-cell staining pattern seen for GIPC and AP-2. The boxed area is enlarged in panels (c-e). In the overlay, GIPC is shown in green, AP-2 is shown in red, and overlap (if present) is yellow. Scale bars = 10mu m.

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