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Figure 5


Fig. 5. FAP133 co-purifies with DHC1b-D1bLIC in sucrose density gradients. (A) Flagellar matrix proteins were separated in a 5 ml 5-20% sucrose density gradient and the resulting fractions were analyzed in two 5-15% polyacrylamide gels and stained with Coomassie Blue (upper panel); similar gels were blotted onto nitrocellulose membrane for immunodetection (lower panels), using antibodies against FAP133, DHC1b, D1bLIC, LC8, IFT139 and FLA10. (B) Fractions 4-7 from a similar gradient (upper panel) were pooled, the sucrose removed and the concentrated sample layered on a separate 5 ml 5-20% sucrose density gradient (lower panel). Immunoblots from both gradients were probed for FAP133 and DHC1b. FAP133 that originally sedimented at ~18 S during the first fractionation shifted to ~10 S in the second gradient suggesting that the FAP133-containing complex had dissociated. (C) Flagellar matrix proteins (Freeze-Thaw; upper panels) or detergent-soluble flagellar membrane and matrix extract (1% Tergitol; lower panels) were fractionated in two separate sucrose-density gradients and analyzed for FAP133 and DHC1b. Detergent treatment caused all DHC1b and FAP133 to migrate more slowly than when obtained by freeze-thaw.





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