Figure 7: Mybbp1a was identified as a novel candidate binding protein for TBP-2.

(a) Purification scheme for the TBP-2 containing protein complex in β-cell nuclear extracts. TBP-2 binding proteins were purified from INS-1 nuclear extracts by using TBP-2 protein-immobilized (TBP-2) or control beads. (b) Identification of the TBP-2 complex. Silver staining was performed. Numbers indicate individual candidate proteins interacting with TBP-2. (c) Eluted proteins were analysed by immuno blotting (IB) for Mybbp1a and TBP-2 antibody. (d) Co-immunoprecipitation analyses. Input (left, 5% lysate) and anti-Myc immunoprecipitates (right, IP:Myc) from HEK293 cells transfected with pCMV-tag2A and pCMV-tag3B vector (−) or FLAG-HA-Mybbp1a and Myc-TBP-2 vector (+) were analysed by immunoblotting (IB) with antibodies to FLAG, Myc and β-actin. The positions for Mybbp1a (p160; p160MBP) and Mybbp1a (p140; p140MBP) (upper), TBP-2 (middle) and β-actin (lower) are shown. (e) Luciferase activity of the UCP-2 -86 enhancer region. INS-1 cells were transfected with PGL4.10 UCP-2 -86 or PGL4.10-luciferase reporter plasmid and each expression plasmid for PGC-1α, Mybbp1a and TBP-2, as indicated. Luciferase reporter activity was normalized by Renilla luciferase activity. (f) A schematic model of TBP-2 function in β-cells. Mybbp1a binds PGC-1α (inactive form) and inhibits UCP-2 transcriptional activity. Induced TBP-2 interacts with Mybbp1a and releases PGC-1α from Mybbp1a, facilitating PGC-1α recruitment on the UCP-2 promoter region. Data are presented as mean±s.d. *P<0.05, **P<0.01, versus control (t-test).