A splice acceptor site mutation affects splicing of IFT81. (a) Gene structure of IFT81 and position of the fla9 mutation. Green box, 5′ UTR; orange boxes, exons; black solid lines, introns; purple box, 3′ UTR; blue arrow, position of the fla9 mutation. The single-nucleotide change from A to G is indicated in red. (b) Alternative splicing of IFT81 in cells grown at 21°C. (i) Representation of multiple IFT81 transcripts between exons 6 and 9. (ii) RT-PCR products of IFT81 between exons 1–4 (top), exons 6–9 (middle), and exons 9–11 (bottom). Individual bands are labelled according to their compositions. (c) Immunoblot of IFT81. Ten micrograms of flagellar proteins isolated from cells grown at 21°C were used in each lane. The same membrane was later probed with an anti-α-tubulin (TUA1) antibody to serve as a loading control. (d) RT-PCR of IFT81 exons 6–9 in various strains grown at 25°C. (e) RT-PCR of DGR14 at both 5′ and 3′ ends of the gene. Amplification of the ribosomal protein gene CRY1 serves as a loading control.