For each Rab column, 5E8 S2 cells were harvested by centrifugation for lysis.

Each of the 23 D. melanogaster Rabs that have a mammalian ortholog were expressed as recombinant GST fusions (in E. coli) bearing the Q>L mutation that is known to stabilize the GTP-bound form (constitutively active). GST-Rab fusions were coupled to glutathione sepharose and incubated with clarified S2 cell lysates (10-20mg/ml protein extract) that were prepared in the absence of any detergent, in the presence of non-hydrolysable GTP analogue GppNHp. Columns were washed and bound proteins were eluted using high salt and GDP. Proteins were concentrated by chloroform/methanol protein precipitation, separated by SDS-PAGE, and trypsin digested for subsequent identification by mass spectrometry.

Purified proteins were identified by liquid chromatography-mass spectrometry. Peptide searches were against FlyBase Dmel 5.9.