Fig. 5.

Inhibition of TBC1D5 function can partially rescue the trafficking defects caused by the VPS35 D620N mutation. (A) HeLa cells expressing GFP-VPS35 D620N were treated with siRNA to silence TBC1D5 expression. After fixation, the cells were labelled with antibodies against Glut1 and the CIMPR. The localisation of Glut1 appears to be shifted away from perinuclear structures after TBC1D5 knockdown. Scale bar: 20 µm. (B) HeLa cells expressing either GFP-VPS35 wild type (WT) or the GFP-VPS35 D620N mutant were treated with siRNA to silence TBC1D5 expression and then labelled with antibodies against Glut and Snx1. The cells were imaged using an automated microscope and the overlap of the Glut1 and Snx1 antibodies measured. There is more overlap of Glut1 with Snx1 in cells expressing VPS35 D620N. The overlap is reduced upon TBC1D5 knockdown but not to levels seen in cells expressing wild-type VPS35. (C) There is no appreciable change in the Snx1 fluorescence following TBC1D5 knockdown. (D) Cells treated as in B were labelled with anti-Glut1 and anti-GM130. (E) There is no appreciable change in GM130 fluorescence after TBC1D5 knockdown. For B-E, values are mean±s.d. and P-values are shown on the graphs.