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Fig. 6. Ninein interacts with ${gamma}$ -tubulin by its N-terminal domain. (A) GFP-ninein expressed for 2 hours in L929 cells recruited the microtubule-nucleation complex (arrowhead) as judged by ${gamma}$ -tubulin (a) or Spc98p (b) staining. The centrosome of a control cell is indicated by an arrow. Bars, 10 µm (the centrosome areas were magnified four times in insets). (B) Fusion constructs including the ninein N-terminal domain immunoprecipitated with myc/ ${gamma}$ -tubulin. Extracts from L929 cells co-expressing myc/ ${gamma}$ -tubulin and either one of the three different GFP-ninein constructs indicated were used for immunoprecipitation experiments with a polyclonal anti-GFP antibody. The weak band of ${gamma}$ -tubulin associated with immunoprecipitation of GFP/Cter-ninein is likely to be non-specific. (C) Two N-terminal domains (residues 1-246 and 1-496) of ninein were expressed as GST fusions and incubated with a lysate prepared from HeLa cells. ${gamma}$ -Tubulin was present in all of the ninein-GST pull-downs. A similar result was obtained for Spc98/GCP3.

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