Fig. 1

Identification of the BBS/CCT complex. (A) Interactions between BBS6, BBS10, and BBS12. Myc- or FLAG-tagged BBS6, BBS10, and BBS12 expression constructs were transfected into HEK293T cells as indicated and lysates were subjected to co-IP assay. (Middle and Bottom) Expression of each component in the lysate. (Top) Amounts of FLAG-tagged proteins coimmunoprecipitated by anti-Myc antibody. IP, immunoprecipitation; IB, immunoblotting. Open arrowhead indicates IgG heavy chain. (B) BBS6, BBS10, and BBS12 associate in vivo. HEK293T cells were transfected with 1 μg of FLAG-BBS12 in one 10-cm dish and immunoprecipitated with anti-FLAG antibody. Co-IP of endogenous BBS6 and BBS10 was probed by Western blotting using antibodies against BBS6 and BBS10. β-Actin was used for normalization of the input. (C) Purification of BBS6 and BBS12 containing protein complexes. Proteins from Myc-BBS6– and FLAG-BBS12–expressing cells (BBS6+12) were purified by sequential affinity purification and analyzed by SDS/PAGE and silver staining. Parental cells were used as a control. Size markers (M) are shown in the left and IgG heavy chain (H) and light chain (L) are marked. (D) Copurification of BBS10 and BBS2 as minor interacting proteins of the BBS/CCT complex. Proteins isolated by sequential purification were resolved in SDS/PAGE and immunoblotted for BBS10 and BBS2. (E) Chaperonin-like BBS proteins form a multisubunit complex with CCT chaperonins. Proteins partially purified by anti-FLAG affinity gel were fractionated by Superose-6 size exclusion chromatography. Elution volumes and approximate molecular weights of fractions where BBS proteins were found were denoted at Top. Void volume was -7.6 mL. Fractions were immunoblotted for Myc (BBS6), FLAG (BBS12), BBS10, BBS7, CCT1, CCT2, and CCT3.