Figure 7. Association of LC1 and tubulin... | Rockefeller University Press

Figure 7.

$Figure 7. Association of LC1 and tubulin within the axoneme. (a) Nitrocellulose blots of axonemal proteins separated in a 5–15% acrylamide gradient gel were incubated in the presence or absence of recombinant LC1 and probed with the R5932 antibody. In the presence of LC1, the tubulin band was recognized, suggesting that LC1 binds tubulin. (left) The blot stained with Reactive brown 10 to detect total protein is shown. (b) Recombinant LC1 was incubated in the presence or absence of taxol-stabilized microtubules and spun in an airfuge for 5 min. Samples were electrophoresed in a 10% acrylamide gel and stained with Coomassie blue. In the absence of microtubules, only a very small fraction of LC1 was found in the pellet. However, upon microtubule addition, considerable LC1 bound microtubules and was present in the pellet fraction. Densitometry based on Coomassie dye binding indicated that the pellet contained a tubulin monomer/LC1 ratio of 1.00:1.06. (c) Axonemes were treated with 0–20 mM EDC, separated in an 8% acrylamide gel, blotted to nitrocellulose, and probed with R5932 and B-5-1-2 antibodies to detect LC1 and α-tubulin, respectively; note that most of the tubulin monomer band was excised from the blot before immunoblotting, as it produces a massive signal that otherwise obscures minor bands. The B-5-1-2 antibody detected two minor tubulin-containing products migrating between the tubulin monomer and dimer bands. The lower of these products precisely comigrated with the LC1-p45 band and was generated with similar kinetics. Numbers in parentheses indicate the approximate molecular mass of the indicated bands (given in kD).$

Association of LC1 and tubulin within the axoneme. (a) Nitrocellulose blots of axonemal proteins separated in a 5–15% acrylamide gradient gel were incubated in the presence or absence of recombinant LC1 and probed with the R5932 antibody. In the presence of LC1, the tubulin band was recognized, suggesting that LC1 binds tubulin. (left) The blot stained with Reactive brown 10 to detect total protein is shown. (b) Recombinant LC1 was incubated in the presence or absence of taxol-stabilized microtubules and spun in an airfuge for 5 min. Samples were electrophoresed in a 10% acrylamide gel and stained with Coomassie blue. In the absence of microtubules, only a very small fraction of LC1 was found in the pellet. However, upon microtubule addition, considerable LC1 bound microtubules and was present in the pellet fraction. Densitometry based on Coomassie dye binding indicated that the pellet contained a tubulin monomer/LC1 ratio of 1.00:1.06. (c) Axonemes were treated with 0–20 mM EDC, separated in an 8% acrylamide gel, blotted to nitrocellulose, and probed with R5932 and B-5-1-2 antibodies to detect LC1 and α-tubulin, respectively; note that most of the tubulin monomer band was excised from the blot before immunoblotting, as it produces a massive signal that otherwise obscures minor bands. The B-5-1-2 antibody detected two minor tubulin-containing products migrating between the tubulin monomer and dimer bands. The lower of these products precisely comigrated with the LC1-p45 band and was generated with similar kinetics. Numbers in parentheses indicate the approximate molecular mass of the indicated bands (given in kD).