The following antibodies were from the indicated vendors: monoclonal mouse anti-acetylated -tubulin (6-11B-1, Sigma-Aldrich) and anti–tubulin (GTU-88, Sigma-Aldrich); monoclonal mouse anti-GFP (JL-8, BD Biosciences); polyclonal rabbit anti-TagRFP (tRFP) antibody (Abdominal233, Evrogen); polyclonal rabbit anti-RFP antibody (PM005, MBL) (referred to as anti-mRFP under Results to distinguish it from anti-tRFP);polyclonal rabbit anti-IFT57 (11083-1-AP, Proteintech) and anti-IFT88 (13967-1-AP, Proteintech); monoclonal mouse anti-actin (C4, EMD Millipore); Alexa Fluor 555-conjugated goat anti-mouse IgG (A21424, Invitrogen); Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, Invitrogen); Alexa Fluor 555-conjugated goat anti-mouse IgG1 (A21127, Invitrogen); Alexa Fluor 647-conjugated goat anti-mouse IgG2b (A21242, Invitrogen); and horseradish peroxidase-conjugated secondary antibodies (115-035-166 and 111-035-144, Jackson ImmunoResearch Laboratories). in earlier models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous manifestation Emiglitate of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to additional IFT-B parts. The detailed connection map Emiglitate as well as assessment of subcellular localization of IFT-B parts between wild-type and Cluap1-deficient cells provides insights into the practical relevance of the architecture of the IFT-B complex. from the pioneering studies of Rosenbaum and colleagues (1). Subsequently, due to the crucial functions for cilia and flagella in various physiological and developmental processes, including cell motility, signaling, and sensory reception, these constructions have been analyzed intensively in metazoans (2,C4). IFT, which techniques numerous proteins bidirectionally between the foundation and tip of cilia/flagella along a microtubule-based structure called the axoneme, is mediated from the large IFT particles with the aid of the anterograde molecular engine kinesin and the retrograde engine dynein. Under high salt conditions, the IFT particle purified from flagella can be divided into two complexes, IFT-A and IFT-B. These complexes are composed of 6 and 14 subunits, respectively, and are thought to connect cargo proteins with molecular motors (4, 5). Mutational analyses in Emiglitate and additional ciliated organisms suggested the IFT-A and IFT-B complexes are primarily involved in retrograde and anterograde ciliary trafficking, respectively. Biochemical studies exposed the approximate architecture of the IFT-A and IFT-B complexes (6,C12), and subsequent studies by Lorentzen and colleagues (13,C15) exposed the structural basis of the relationships among several IFT-B subunits. The IFT-B complex consists of the core subcomplex, including at least nine subunits (IFT88, -81, -74, -70, -52, -46, -27, -25, and -22) and at least five peripherally connected proteins (IFT172, -80, -57, -54, and -20) (examined in Refs. 4 and 5). Even though IFT-B subunits are evolutionarily conserved (2, 16), the architectures of the IFT-B complex in additional ciliated organisms, including mammals, remain poorly understood. Furthermore, it is also unclear how the peripherally connected proteins are integrated into the full IFT-B complex. Recently, we developed a novel technique, the visible immunoprecipitation (VIP) assay, as a method for studying protein-protein relationships and used it to determine NT5E the architectures of two multisubunit complexes, the BBSome and exocyst (17), both of which consist of eight subunits and have been implicated in protein trafficking to and/or within cilia. The VIP assay can visually detect binary protein relationships under a conventional fluorescence microscope without the necessity of electrophoresis and immunoblotting. Furthermore, the assay can determine relationships between more than two proteins at a time, including one-to-many and many-to-many protein relationships (17). In this study, we applied the VIP assay to delineate the architecture of the mammalian IFT-B complex. The results exposed that the complex consists of 16 subunits and may be divided into core and peripheral subcomplexes comprising 10 and 6 subunits, respectively. In particular, our data unequivocally showed that TTC26 and Cluap1, both of which have been referred to Emiglitate as IFT-B accessory proteins or candidate IFT-B subunits in earlier review content articles (2, 4, 18), are integral components of the core and peripheral subcomplexes, respectively. Furthermore, our findings reveal how the six peripheral subunits interact with one another to constitute the peripheral subcomplex. Materials and Methods Plasmids The full coding sequences of IFT-B proteins outlined in supplemental Table S1 were cloned into numerous fluorescent protein vectors, as demonstrated in supplemental Table S2. Antibodies and Reagents Preparation of polyclonal rabbit anti-Cluap1 antibody was explained previously (19). The following.
