Figure 8.

A, Levels of Orbit protein are greatly reduced in orbit mutants. Equivalent amounts of total protein extract corresponding to three wild-type ovaries (lane 1); ten orbit/orbit ovaries (lane 2); ten wild-type larval brains (lane 3); and 20 orbit ³ /Df(3L)orbit² larval brains (lane 4), were blotted and detected with an affinity-purified Orbit antibody. The antibody recognizes a major band of 165 kD that is reduced in amount in extracts from orbit ovaries and is barely detectable in extracts of orbit larval brains. B, Orbit copurifies with microtubules. Western blot of the fractions obtained during the purification of microtubules from wild-type embryos (see Materials and Methods). The blot was probed with anti-Orbit (diluted 1:1,500) and antitubulin (diluted 1:4) antibodies. Lane 1, 20 μg of crude embryonic protein extract; lane 2, 20 μg of protein from the supernatant fraction after the centrifugation through sucrose; lane 3, 20 μg of proteins removed from the microtubule pellet by a 250 mM NaCl wash; and lane 4, 10 μg of the final microtubule fraction. C and D, Orbit binds to microtubules in the presence of GTP, but not its nonhydrolyzable analogue GTP-γ-S. Recombinant Asp (lane 1), Orbit protein (lane 2), and BSA (lane 3) were transferred to PVDF membranes, incubated with the indicated nucleotides, and subsequently polymerized microtubules. Binding of microtubules was assessed using anti-β-tubulin (see Materials and Methods). E, Orbit binds to microtubules in solution in the presence of GTP (I), but not GDP (II) or GTP-γ-S (III). Soluble Orbit protein was incubated with different concentrations of microtubules (as indicated on the figure) in the presence of GTP (I), GDP (II) or GTP-γ-S (III). Polymerized microtubules were recovered by centrifugation. Presence of Orbit in the microtubule pellet and the supernatant was assessed by immunoblot using anti-Orbit antibody.