Figure 3.

Covalent modification of tubulin by IAABE. (A) Tubulin or BSA were incubated in the presence of ³H-IAABE for 0 (▲ tubulin) or 60 minutes (■ tubulin; ♦ BSA). Samples were separated on a polyacrylamide gel and blotted onto a nitrocellulose membrane. Lanes were dissected into slices and counted for radioactivity. Tubulin has a molecular weight of 55 kDa and BSA 68 kDa. The peak at 55 kDa is monomeric tubulin and the peak at 120 kDa represents tubulin dimers. (B) CEM cells were incubated in the presence of 0.37 µM ³H-IAABE for 0 (•), 1 (■), 4 (▲) and 12 hours (♦). Cells were harvested and lysed directly into gel loading buffer. Samples were separated on a polyacrylamide gel and blotted onto a nitrocellulose membrane. Lanes were dissected into slices and counted for radioactivity. Note major peak at 55 kDa. (C) DEAE purification of ³H-IAABE-labeled tubulin from CEM cells. Extracts from ³H-IAABE-treated cells (1 hour incubation) were purified with the DEAE method. Bound tubulin (♦) and unbound fractions (■) were blotted and counted as described above. Note that 95% of radioactivity co-eluted with the tubulin.