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Fig. 8. Depletion of Rab27a and MyoVa, but not EB1, prevents Mlph-mediated rescue of melan-ln melanosome transport defects. Melan-ln melanocytes were sequentially transfected first with siRNA to allow depletion of the indicated proteins and second with plasmid DNA allowing expression of Myc-tagged Mlph as described in Materials and Methods. Cells were then stained with antibodies allowing detection of the overexpressed Myc-Mlph (panels A,C,G,K) and siRNA-targeted protein to confirm depletion in each cells (panels D,H,L). Panels E, I and M are merged fluorescent images showing Myc-Mlph (green) and siRNA-targeted protein (red) and B, F, J and N are transmission images showing the distribution of melanosomes. Arrows indicate colocalisation of Mlph protein with the cytoplasmic surface of melanosomes. Panel O shows quantification of the extent of rescue of melanosome transport by the Mlph in melan-ln cells siRNA-depleted of Mlph-interacting proteins using the indicated oligonucleotides. Rescue efficiency for populations of transfected cells was determined as described in Hume et al. (Hume et al., 2006). Shaded boxes indicate 25th/75th percentile; solid and dotted bars within boxes indicate median and mean values for each population of cells; outer bars indicate 5th/95th percentile; and outer points indicate outliers. The statistical significance of rescue measurements for populations of transfected cells relative to positive and negative controls is shown in Table 2. Bar, 20 µm.
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