FIGURE 7:

TfR trafficking is not significantly perturbed by mVps45 knockdown. (A) Data from a representative experiment assaying levels of cell surface Texas red Tf binding under basal or insulin-stimulated (100 nM for 30 min) conditions in control or mVps45-knockdown cells. Data are presented as percentage of the signal observed in basal control cells; each point is the mean of triplicate determinations at each condition. The experiment was repeated a further two occasions with similar results. The lower cell surface binding values observed in knockdown cells were significant in all experiments. (B) Recycling of Texas red Tf in control (open circles) and mVps45-knockdown (filled circles) cells. Briefly, cells were incubated with Texas red Tf for 3 h, surface-associated Tf was removed by a brief acid wash, and then cells were incubated in recycle buffer for the times shown. At each time, the Texas red Tf released into the media and that remaining cell associated were quantified in quadruplicate. The data from a typical experiment of this type are shown, in which the logarithm of the cell-associated Tf is plotted as a function of time. Inset, immunoblots of lysates of the same cells probed with anti-mVps45 or anti-TfR. Four experiments of this type revealed no statistically significant difference between the two conditions.