Supplementary Figure 4: UBXN10 interacts with the Intraflagellar Transport B (IFT-B) complex that controls anterograde trafficking into cilia.
(a) HA-UBXN10 and MYC-IFT-B were in vitro translated in HeLa lysates. α-MYC immunoprecipitates were resolved by SDS-PAGE and probed for associated HA-UBXN10. GFP was used as a negative control. UNC13B, which is not an IFT-B subunit, was also validated in this manner. (b) A subset of IFT-B proteins are present in the HeLa IVT lysate as determined by immunoblotting. (c) MYC-IFT25 was in vitro translated in HeLa IVT lysate and immunoprecipitated with MYC agarose. Bound complexes were analyzed by LC-MS/MS. IFT-B proteins in HeLa lysate that interacted with MYC-IFT25 are shown. (d) MYC-GFP, IFT25 and IFT27 were in vitro translated in HeLa IVT lysate and immunoprecipitated with MYC agarose. Immunoprecipitates were probed for IFT27. MYC-IFT27 binds IFT25 in HeLa lysate. (e) UBXN10 binds specifically to the CLUAP1 subunit of IFT-B. 35S-labelled-IFT-B subunits were translated in rabbit reticulocyte lysates and incubated with GST alone or GST-UBXN10 or GST-VCP purified from E.Coli. GST pull-downs were resolved by SDS-PAGE, stained with Coomassie and subjected to autoradiography. ∗: 35S-labelled- IFT52 was inadvertently loaded twice in the input gel alone. The longer exposure of the autoradiograph shows weaker (non-specific) binding to IFT46, 52 and 57 by GST, GST-UBXN10 and GST-VCP.
