Expression of a GDP-locked and constitutively inactive Rab7a (T22N) results in reduced endosomal retromer CSC that is similar to what is observed upon overexpression of wild-type TBC1D5 (Seaman et al., 2009). Thus, we next tested whether loss of TBC1D5 function could enhance levels of retromer CSC in cells stably expressing the Rab7aT22N mutant by silencing TBC1D5 expression in a panel of seven different cell lines expressing GFP-tagged proteins. In Fig. 2C, loss of TBC1D5 expression increases the VPS26 fluorescence intensity across all the cell lines tested, but in cells expressing GFP-Rab7a, GFP-Rab5 or GFP-Rab9, the increase was not statistically significant. There was, however, a statistically significant increase in cells expressing the GDP-locked Rab7aT22N mutant, most likely due to increased activation of endogenous Rab7a that is present in the cell line. Similarly, the cells expressing the constitutively active Rab7a Q67L mutant also demonstrated an increase in endosomal VPS26 after TBC1D5 knockdown, as these cells contain endogenous Rab7a in addition to the stably expressed GFP-tagged version. Knockdown of Rab7a reduced the VPS26 fluorescence intensity, except for experiments where Rab7a was expressed as
