or VPS26 (Fig. 1B). Loss of TBC1D5 appears to lead to somewhat brighter staining of VPS35 and VPS26 (asterisks). To more quantitatively investigate the effect of loss of TBC1D5 on the membrane association of the retromer CSC, we used automated microscopy as in our previous study in which the role of Rab7a and Snx3 in regulating recruitment of the CSC was reported (Vardarajan et al., 2012; see also Breusegem and Seaman, 2014). This approach enables the imaging of hundreds of cells whilst avoiding unintended bias that may occur through manual imaging. Control cells and cells treated to silence TBC1D5 expression were labelled with antibodies against VPS26, TBC1D5 the CIMPR and TGN46. The cells were imaged using an automated microscope and the fluorescence intensity quantified (Fig. 1C). Loss of TBC1D5 expression increases the total intensity of VPS26 staining by ∼40% and is statistically significant (P-values are shown in the figure legend). The fluorescence intensity of the CIMPR also increased, but the fluorescence intensity of TGN46 was only marginally increased. A gain in total intensity could be due to increased signal per spot/endosome, or more spots. Therefore, we also plotted the average number of spots per cell (Fig. 1D). There was a
