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Fig. 5. Star cofractionates with dynein. Shown are immunoblot results using antibodies against Star-HA and dynein heavy chain (DHC). (A) A crude preparation of vesicles (V) derived from fly heads expressing HA-tagged Star (see Materials and Methods) was centrifuged at 100,000 g and the resulting supernatant (S) and pellet (P) fractions were analyzed by immunoblot. Equivalent volumes were loaded in each lane. (B) A membrane-enriched sample was fractionated on a Nycodenz step gradient. Equal volumes of each fraction were analyzed by western blot (fraction 1=bottom of gradient). The starting sample (L) and the pellet resulting from the gradient centrifugation (P) are also shown. Star and Dhc are present in overlapping fractions. (C) A vesicle membrane pellet (see part A) from hsStar-HA flies was resuspended in soluble (100,000 g) extract derived from wild-type flies. Paclitaxel (taxol) was used to promote microtubule polymerization in the absence (–) or presence (+) of ATP. In control samples, where no microtubules were assembled (–taxol, –ATP), neither dynein nor Star-HA is present in the pellet following low-speed centrifugation. In the pellets containing polymerized microtubules, Star-HA shows a greater enrichment in the absence of ATP, consistent with an interaction with dynein. The immunoblot shows pellets resulting from each experimental condition. (D) Membrane-enriched samples containing both dynein and Star-HA were prepared from S2 cultured cells by flotation on step gradients, and proteins were crosslinked with EDC for the times listed above each lane. As time progresses, increasing amounts of a very high molecular mass complex containing Dhc are detected at the top of the gel (arrowhead), with a corresponding decrease in noncrosslinked Dhc (arrow). Similarly, on a replicate blot, increasing amounts of Star-HA are seen in a very high molecular mass band (arrowhead) that coincides with the crosslinked Dhc band, while noncrosslinked Star-HA (arrow) decreases over time. Tubulin and actin, shown as negative controls, do not enter the high molecular mass complex. Note the formation of dimeric tubulin over time (
110 kDa; asterisk).
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