Figure 4.

p61 migrates anomalously in SDS-polyacrylamide gels. (a) Twenty micrograms of the MBP-p61(7-586) fusion protein was electrophoresed in a 5–15% acrylamide gradient gel, both before and after incubation with factor Xa, and stained with Coomassie Blue. The intact fusion protein has a calculated mass of 103,130 Da but migrated with M_r ∼135,000. The minor bands derive from cleavage of MBP-p61(7-586) by endogenous bacterial proteases. After factor Xa cleavage of the fusion protein, MBP migrated at ∼M_r 40,000 as observed previously, whereas the p61(7-586) segment (calculated mass 60,662 Da) had M_r ∼100,000. (b) Wild-type axonemes (∼100 μg) prepared in the presence of a protease inhibitor cocktail were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie Blue (left) or blotted and probed with the CT220 antibody (right). Two prominent bands of M_r102,000 and 40,000 were obtained. The top band corresponds to p61, which migrates anomalously. The diffuse band located above tubulin is a proteolytic fragment of p61.