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. 2017 Feb 1;28(3):429–439. doi: 10.1091/mbc.E16-11-0813

FIGURE 2:

FIGURE 2:

One-to-many subunit interactions in the IFT-A complex. (A, B) Interaction of IFT140 with IFT122–IFT144. HEK293T cells cultured in 6-cm dishes were transfected with expression vectors for EGFP-IFT140 and mChe-fused IFT-A subunits as indicated. (A) Lysates prepared from the cells were precipitated with GST-tagged anti–GFP Nb prebound to glutathione–Sepharose beads and processed for the VIP assay. (B) Proteins bound to the precipitated beads (top two panels) or input proteins (bottom two panels) were subjected to immunoblotting with an anti-RFP antibody (top and third panels) or anti-GFP antibody (second and bottom panels). (C, D) Interaction of TULP3 with the IFT122–IFT140–IFT144 trimer. Lysates prepared from HEK293T cells cotransfected with expression vectors for EGFP-TULP3 and mChe-fused IFT-A subunits, as indicated, were subjected to the VIP assay (C) and immunoblotting (D) as described. (E, F) Interaction of IFT139 with IFT43–IFT121. Lysates prepared from HEK293T cells coexpressing EGFP-IFT139, HA-IFT122, and mChe-fused IFT-A subunits, as indicated, were subjected to the VIP assay (E) and immunoblotting (F). To distinguish between IFT121 and IFT122, different tags, namely, mChe and HA, were used. (G, H) Interaction of IFT122 with IFT43–IFT121. Lysates prepared from HEK293T cells coexpressing EGFP-IFT122 and mChe-fused IFT-A subunits, as indicated, were subjected to the VIP assay (G) and immunoblotting (H).

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