JCS045096 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Figure S1 -

  Fig. S1. Molecular size assessment of DHC3 by SDS-PAGE. (A) Immunoblot analysis of DHC3, DHC4, DHC5 and DHC11 using SDS-PAGE without urea. The far right three lanes show silver-stained SDS-PAGE patterns of oda1 axonemes before and after UV irradiation, and the dynein f fraction purified by HPLC, respectively. Proteins were separated using a 5% acrylamide gel without urea. Each antibody detected a band in the DHC region (filled arrowhead) in samples untreated with UV irradiation. Note that the DHC3 band position is much higher than the DHC1 (fα DHC of dynein f/I1) band, as was observed in the 3-5% gradient gel with urea (Fig. 3A). The apparent molecular weight of DHC3 is approximately 100 kDa larger than DHC1 (535 kD), the largest among all previously known axonemal DHCs in Chlamydomonas. (B) Domain structures predicted in a conventional DHC and DHC3. Schematic diagram of the DHC9 sequence and the predicted DHC3 sequence registered in the Chlamydomonas genome database. The AAA, stalk and C-terminal domains are indicated in different colors. A tall red box flanked by a pair of blue boxes represents the microtubule-binding stalk. DHC3 is larger than DHC9 by ∼1200 amino acids. Arrowheads show amino acid sequences that are present in DHC3 but absent in DHC9.

• Supplemental Figure S2 -

  Fig. S2. Specificity of affinity-purified DHC5, DHC9, and DHC11 antibodies. (Left) Coomassie brilliant blue (CBB)-stained SDS-PAGE (5-20% acrylamide gradient) of oda1 axonemes. (Right) Immunoblot analysis of oda1 axonemes with affinity-purified DHC5, DHC9 and DHC11 antibodies.

• Supplemental Figure S3 -

  Fig. S3. Localization length of DHC11. A histogram of the lengths of the proximal axoneme region where the DHC11 signal was strong (observation in 22 axonemes). The average length ± s.d. is 2.1±0.3 µm.

• Supplemental Figure S4 -

  Fig. S4. Change in the relative content of DHC4 and DHC11 during flagellar growth. (Upper panel) SDS-PAGE analysis of axonemes prepared at the indicated time points following flagellar amputation. Equal amounts of axonemal proteins (10 µg) were loaded in each lane, and the gel was stained with SyproRuby. Only the high molecular weight region around the DHC bands is shown. The DHC4 and DHC11 bands were strongest 30 minutes after flagellar amputation. The DHC band (DHC5 and DHC9) in which dynein b and c overlap showed a slight reduction in intensity 30 minutes following flagellar amputation. The DHC1 band (fα DHC of dynein f/I1) staining intensity is almost constant throughout flagellar growth. (Lower panel) Quantification of changes in relative DHC content during flagellar growth.