Figure 8.

The Dhc10 antibody identifies the 1β Dhc as the gene product of the IDA2/Dhc10 locus. (A) Specificity of I1 Dhc antibodies. The I1 complex was purified by sucrose density gradient centrifugation and analyzed on 5% polyacrylamide gels to resolve the 1α and 1β Dhcs (left lane). Duplicate immunoblots were probed with an affinity-purified Dhc1 antibody (middle lane) and an affinity-purified Dhc10 antibody (right lane). (B) Reassembly of I1 complex subunits in Dhc10 transformants. Axonemes were isolated from wild-type and mutant strains, run on 5% polyacrylamide gels, and blotted to either nitrocellulose or polyvinylidene difluoride. One strip containing only the Dhc region was probed with the Dhc1 antibody to detect the 1α Dhc (top blot). A second strip containing the region above 85 kDa was probed with the Dhc10 antibody to detect the 1β Dhc and related N-terminal fragments (middle blot). A third strip containing the 140-kDa region was probed with the IC140 antibody (bottom blot). ida2-6 and ida2-7 are two different insertional mutants (see Figure 5A). Both strains were cotransformed with Dhc10 constructs encoding N-terminal 1β Dhc fragments of varying lengths (see Figure 5, B and E). Note that the 1β Dhc fragment encoded by the pCAP3 subclone does not contain the peptide epitope used to generate the Dhc10 antibody, so the ∼92-kDa fragment cannot be detected by Western blot analysis of axoneme polypeptides.