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 chemical crosslinker, VPS26 was recovered from lysates by immunoprecipitation. The immunoprecipitates were analysed by mass spectrometry and the ratio of peptides detected that contained ‘heavy’ or ‘light’ amino acids was calculated. Fig. 3B shows the results obtained from three different cell lines. In each instance, the TBC1D5 heavy:light ratio is markedly above 1, indicating loss of protein from the light fraction consistent with the knockdown of TBC1D5. The ratios for the retromer CSC protein were all close to 1, which is to be expected. Most retromer-associated proteins, for example, those of the WASH complex (strumpellin, KIAA1033 and Fam21) generated ratios in the range of 0.7-0.5, indicating increased detection of those proteins in the TBC1D5-knockdown samples.
