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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.
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