Figure 3

The BTB domain-containing proteins bind LRRs, while the ΔBTB mutant enhances the binding interaction. A. Ctb73 contains three protein-protein interaction domains; BTB domain, BACK domain and PHR (kelch) domain. B. MYC-Ctb73 was immunoprecipitated and probed with anti-HA antibodies to detect binding to LRR1 and LRR5 (upper panel). Lower panels show the expression levels of the HA-LRRs in transfected cells. C. MYC-Ctb73 wild-type or deletion mutants were pulled down by anti-MYC antibody and probed against HA (upper panel) to locate LRR5 binding site on Ctb73. Relative expression levels of HA-LRR5 are shown in lower panel. D. Two sequential immunoprecipitations were performed on cell lysates expressing FLAG-TEV-Cul3, MYC-Ctb73 and HA-LRR5. FLAG beads brought down Cul3 complexes that were cleaved off the resin using TEV protease. The eluted proteins were verified by Western blot for Cul3. Subsequent immunopreciptiation with MYC antibody brought down a protein complex containing Ctb73. Western blot analysis showed LRR5 binding to the Cul3-Ctb73 complex. Relative protein levels are shown in bottom three panels. E. Cell lysates co-expressing Cul3, Ctb73 and LRRs were used in the immunoprecipitation of FLAG-Cul3 that pulled down HA-LRR1 and HA-LRR5. In the same transfection set, immunoprecipitation of FLAG-Cul3 also brought down MYC-Ctb73. Relative expression levels of LRR1, LRR5 and MYC-Ctb73 were verified by Western blotting. See also Additional file1: Figure S1.