As Rab7a associates with the retromer CSC in a GTP-dependent manner (Harrison et al., 2014; Priya et al., 2015), we investigated whether increased Rab7a-GTP would enhance the association of Rab7a with the retromer CSC by silencing of TBC1D5. The results of a native immunoprecipitation of GFP-tagged Rab7a are shown in Fig. 3A. The retromer CSC proteins VPS35 and VPS26 co-immunoprecipitated with GFP-Rab7a, but not GFP-Rab5, and the levels of the retromer CSC proteins were increased in TBC1D5-knockdown cells. The elevated levels of retromer CSC on the endosomal membrane would be predicted to enhance the association of the retromer CSC with proteins that it is known to interact with on endosomes. Therefore, a protocol for stable isotope labelling with amino acids in cell culture (SILAC) was employed and cells were labelled with amino acids synthesised with either heavy or light isotopes of carbon and nitrogen over a number of passages to ensure that the amino acids were incorporated into the cellular proteins. TBC1D5 expression was silenced by RNAi in cells labelled with ‘light’ amino acids and, following treatment with a membrane-permeable
