If loss of TBC1D5 function is enhancing the endosomal localisation of the retromer CSC by activating Rab7a, it should be possible to detect elevated levels of active Rab7a. Therefore, cells expressing GFP-Rab7a were treated with siRNA to ablate TBC1D5 expression. Control and knockdown cells were labelled with antibodies that specifically recognise GTP-bound Rab7a. The signal from the anti-Rab7a-GTP antibody was low in control cells (Fig. 2A, top) but markedly enhanced in cells where TBC1D5 expression had been abolished (bottom). The increase in fluorescence intensity of the Rab7a-GTP signal was quantified using automated microscopy for cells expressing a range of GFP-tagged proteins (Fig. 2B). Only where GFP-Rab7a is expressed could a signal for the anti-Rab7a-GTP antibody be detected and loss of TBC1D5 expression resulted in a significant increase in that signal (nearly two-fold for cells expressing GFP-Rab7a).
