Properties of LC1–tubulin interactions in situ. (a) Axonemes from strains expressing mutant myc-tagged forms of LC1 were treated with 0 or 5 mM EDC electrophoresed and immunoblotted using the 9E10 monoclonal antibody against the myc epitope or R5932 to detect LC1. In all strains examined, myc-tagged LC1 was readily incorporated into the M_r 66,000 LC1–tubulin cross-linked product, indicating that these proteins remain in direct contact. Furthermore, there were no obvious differences in the rate of incorporation of the tagged mutant forms compared with wild type, as both LC1 and myc-LC1 bands were reduced with similar kinetics. (b) Axonemes were treated with either 0 or 20 mM EDC in buffer or in the presence of 1 mM ATP, 1 mM ADP, 1 mM ATP plus 100 µM vanadate, and 100 µM vanadate alone. Samples were electrophoresed in a 5–15% acrylamide gradient gel, blotted to nitrocellulose, and probed with the R5932 antibody. The M_r 66,000 LC1–tubulin cross-linked product was generated by EDC treatment in similar yield under all nucleotide conditions, suggesting that LC1 and tubulin are permanently tethered to each other during the mechanochemical cycle. Several of the higher order products (arrows) show mass increase in steps of ∼50 kD and likely derive from cross-linking of the LC1–tubulin product to ≥1 additional tubulin molecules. (c) To test whether LC1–tubulin interactions were modified in a Ca²⁺-dependent manner, axonemes were resuspended in buffer containing 1 mM Ca²⁺ or EGTA in the presence or absence of 1 mM ATP/100 µM vanadate and were subsequently treated with 0 or 5 mM EDC. Samples were electrophoresed in a 10% acrylamide gel and stained with Coomassie blue (top) or probed with the R5932 antibody against LC1 (bottom). No differences in the yield of the LC1–tubulin product were observed as a consequence of Ca²⁺ addition.