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. 2010 Apr 19;189(2):223–232. doi: 10.1083/jcb.200911018

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© 2010 Hsu et al.

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Figure 1. — Screen of a Rab GAP library identifies the TBC1D10 family as regulators of exosome secretion. (A and B) PLP-EGFP was coexpressed with a library of 38 different EGFP-tagged wild-type (WT) Rab GAPs in Oli-neu cells using a plasmid ratio of 2:1 (EGFP-TBC/PLP-EGFP). Cells transfected with both PLP-EFP and YFP were used as a reference. 16 h after transfections, the cells were switched to serum-free medium, and the medium was collected after ∼4 h of further incubation before submitting it to sequential centrifugation steps. The resulting 100,000 g pellets (exosome fraction) of each centrifugation step were analyzed by Western blotting for PLP-EGFP and the EGFP-TBCs (Rab GAPs) by anti-GFP antibodies. The mean of three independent experiments is shown in the graph. Those Rab GAPs that reduced exosome release of PLP below 60% of the control (boxed), were compared with their catalytically inactive arginine to alanine (RA) mutants, and the zoomed-in graph is shown in B. Error bars indicate the SD. (C) Western blot of the cell lysates (CL) and 100,000 g pellets (P100) is shown for one representative experiment with TBC1D10.