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Figure 8


Fig. 8. Association of CrLis1 with ODA IC2 and rat NudC. (A) GST pull-down assay of Chlamydomonas flagellar membrane plus matrix extract. The extract (input 5%) was mixed with glutathione beads coated with GST-CrLis1 or GST and bound proteins were recovered by centrifugation and analyzed by SDS-PAGE and western blotting using antibodies as indicated. IC2 of the ODA complex cosediments with GST-CrLis1 beads, whereas {alpha}-tubulin does not. (B) GST pull-down assay of Chlamydomonas flagellar extract showing that CrLis1 and IC2 cosediment with GST-rNudC. (C) Coomassie-Blue-stained gel of the pellet fractions shown in panels A and B. (D,E) GST pull-down assay of rat brain extract (input 15%). Panel D shows a western blot analysis of the input and pellet fractions, panel E shows the corresponding Coomassie-Blue-stained gel. Note that native rNudC cosediments with GST-CrLis1. (F,G) GST pull-down assay showing direct association between CrLis1 and rNudC. In panel F, purified recombinant MBP-CrLis1 (CrLis1) or MBP was mixed with glutathione beads coated with GST-rNudC and bound proteins were recovered by centrifugation. The left-hand panel shows a Coomassie-Blue-stained gel of the pellet (pel) and supernatant (sup) fractions. Note the presence of MBP-CrLis1 (asterisk) in both the supernatant and pellet fractions. The filled circle corresponds to GST-rNudC, which is present in equal amounts in the two pellet lanes. In the middle and right-hand panels, corresponding western blot analyses of the pellet fractions are shown. MBP-CrLis1 cosediments with GST-rNudC whereas MBP does not cosediment. (G) Control experiment in which purified MBP-CrLis1 (input 10%) was mixed with GST-rNudC- or GST-coated beads, subjected to centrifugation and analyzed by SDS-PAGE and immunoblotting. MBP-CrLis1 cosediments with GST-rNudC, but not with GST alone. Asterisks mark the MBP-CrLis1 that is cosedimenting with the GST-rNudC beads.





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