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Figure 3


Fig. 3. Confirmation that the MS analysis identified the correct RSPs. (A) Western blot showing that antibodies based on the sequences predicted for RSPs 7, 8, 9, 10, 11 and 12 recognize proteins present in wild-type (WT) axonemes but absent in axonemes from the spokeless mutant pf14. (B) Western blot of sucrose density gradient fractions showing that an antibody based on the sequence predicted for RSP11 recognizes a protein that co-sediments with solubilized 20S WT radial spokes (upper panel) or 15S spoke stalks from the spoke-head-less mutant pf17 (lower panel). The blot was also probed with an antibody to RSP16 (Yang et al., 2005) as a marker for the 20S spokes and 15S spoke stalks. Similar results were obtained for RSP7 (data not shown). (C) Antibodies raised against RSPs 1 and 5 purified from spots on 2D gels recognize the recombinant proteins (R-RSP1 and R-RSP5). In addition, the recombinant proteins, which are His tagged, migrate at the expected size relative to the native proteins in WT axonemes. The proteins are absent in pf14 axonemes.





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