expressing the VPS35 D620N mutant were treated with siRNA to silence TBC1D5 expression. In Fig. 5A, cells expressing the VPS35 D620N mutant were treated with siRNA to silence TBC1D5 expression. We observed that loss of TBC1D5 appears to reduce the extent of Glut1 localisation to the perinuclear compartment that was observed in control cells. To quantify this change on Glut1 localisation, cells expressing either wild-type GFP-VPS35 or the D620N mutant were treated with siRNA to abolish TBC1D5 expression. After fixation, the cells were stained with antibodies against Glut1 and either Snx1 or the Golgi marker protein GM130 and then imaged using an automated microscope. In Fig. 5B, the expression of the D620N mutant causes increased colocalisation of Glut1 with Snx1 relative to cells expressing wild-type VPS35. The fraction of Glut1 fluorescence that colocalises with Snx1 is reduced after loss of TBC1D5 expression. A similar result was obtained for Glut1 and GM130 (see Fig. 5D) and both sets of observations are statistically significant. The reduction in the fraction of Glut1 that colocalises with intracellular markers such as Snx1 or GM130 is consistent with increased localisation of Glut1 to the cell surface, although there does not appear to be a full rescue
