Fig. 3. Numb binds to transitin. (A) Structure of the chick transitin protein and of its deletion mutants. (B) Upper panel shows the binding of Numb that was extracted from chick Numb-transfected NIH3T3 cells to one GST-fused transitin deletion protein (328-816), but not to bacterially expressed GST (containing His tags and an S tag, see Materials and methods) or to the other GST-fusion of transitin deletions that were blotted. Bound Numb is detected by anti-Numb antibody. Lower panel shows Coomassie Brilliant Blue (CBB) protein staining of SDS-PAGE gel. Positions of recombinant proteins are indicated by asterisks and a square bracket. (C) Structure of the chick Numb protein and of its deletion mutants. (D) Direct binding of Numb to Trans(328-816). Whole extracts of MBP- and MBP-Trans(328-816)-fusion expressing bacteria were separated on SDS-PAGE, blotted on a membrane and incubated with purified GST-fusions of Numb. Bound GST-Numb fusions were detected by anti-GST antibody. Expression of MBP and MBP-Trans(328-816)-fusion was confirmed by anti-MBP antibody. (E) Transitin and Numb bind directly in a liquid-phase assay. Purified MBP-Trans(328-816) and GST-Numb(158-582) were mixed and pulled down by anti-GST antibody. Immunoprecipitated (IP) MBP-Trans(328-816) was detected by western blotting (WB) with anti-MBP antibody. MBP-Trans(328-816) used for this assay is indicated as `input' in the left panel, detected by western blotting with anti-MBP antibody. IP, immunoprecipitated; PTB, phospho-tyrosine-binding domain; WB, western blotting.