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Fig. 7. Dominant-negative XStau blocks VLE localization in vivo. (A) Full-length
XStau (WT, above) and XStau234 (DN, below) are shown. The double-stranded RNA
binding domains (dsRBDs) are shown in blue and are numbered, and the
tubulin-binding domain (TBD) is shown in white. (B) RNA encoding either
XStau234 (DN, lanes 1, 3) or full-length XStau (WT, lane 2) was injected into
stage III oocytes, and S10 lysates were prepared after 16 hour incubation.
Expression of injected RNA was analyzed by immunoblotting with FLAG antibodies
(lanes 1, 2); immunoblotting with XStau antibodies (lane 3) was used to
compare expression of XStau234 (DN, below) to that of endogenous XStau
(above). (C) Fluorescently labeled VLE RNA was injected into oocytes
expressing either full-length XStau (WT, left) or XStau234 (DN, right).
Examples are shown of the predominant phenotypes observed for oocytes
expressing full-length XStau (WT), showing localization of VLE RNA (red) and
XStau234 (DN), exhibiting no detectable localization. Scale bars: 100 µm.
(D) Localization was scored for VLE-injected oocytes expressing either WT
XStau (left, n=151) or DN XStau (right, n=117). The graph
shows the percentage of oocytes that exhibited strong vegetal localization
(green), weak vegetal localization (yellow) or no detectable localization
(red). (E) S10 lysates were prepared from oocytes expressing either XStau234
(DN, lane 1) or full-length XStau (WT, lane 2), and immunoprecipitation was
performed using FLAG antibodies. Bound proteins were separated by SDS-PAGE and
immunoblotted with Vg1RBP/vera antibodies (above) and hnRNP I antibodies
(below).
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