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Fig. 6. The VAB-1 tyrosine kinase domain physically interacts with the
juxtamembrane and CC1 region of SAX-3/Robo. (A) Schematic diagrams of the
VAB-1 Eph RTK and SAX-3/Robo receptor. VAB-1 Kinase region (669 aa-985 aa) was
fused in frame to the GAL-4 DNA-binding domain and used as bait in yeast
two-hybrid assays. The SAX-3 intracellular CC1, CC3 and CC2 regions are shown
as blue boxes. SAX-3 does not have a recognizable CC0 consensus and CC3 is N
terminal to CC2 when compared with other Robo receptors. (B) Deletion
constructs of the intracellular portion of SAX-3 were tested for their ability
to bind the VAB-1 tyrosine kinase domain. Liquid ß-Galalctosidase units
and yeast X-GAL overlay assays indicate the quantity of activation
(interaction). The juxtamembrane and CC1 region is necessary and sufficient
for the interaction with the VAB-1 kinase region (dashed rectangle). (C) GST
`pull-down' experiments confirm that the SAX-3 juxtamembrane and CC1 region
(900 aa-1030 aa) is sufficient for the interaction with the VAB-1
intracellular region. MBP-SAX-3 E. coli lysates were incubated with
GST or GST-VAB-1. MBP-SAX-3 input (Load), unbound (UB) and bound (B) fractions
were analyzed by SDS-PAGE and western blotting using anti-MBP to visualize
SAX-3. The SAX-3 interaction is specific to GST-VAB-1 and not GST alone.
GST-VAB-1 is also specific for MBP-SAX-3, as it does not bind the MBP
degradation products (asterisks) in the Load.
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