FIGURE 3.

Tubulin degradation is induced by ITCs at post-synthesis level. A, tubulin-binding agents have no effects on tubulin levels in cells. HeLa cells were treated with 1, 10, and 100 nm and 1 and 10 μm vinblastine or colchicine for 8 h. The whole cell lysate was extracted and blotted for α- and β-tubulins. B, ITC-induced tubulin degradation is initiated by aggregation and is irreversible. HeLa cells were treated with 10 μm BITC for ½ and 1–4 h before medium containing BITC was replaced, and cells were grown in fresh medium up to 6 h. The soluble fraction and the whole cell lysate were extracted and blotted for α-tubulin. C, ITC treatment does not alter tubulin transcripts. HeLa cells were treated with 10 μm BITC for 2, 4, 8, and 24 h. Total RNA was isolated and used as template for one-step RT-PCR of both α- and β-tubulins. PCR products after 25 cycles were visualized on a 1.5% agarose gel containing ethidium bromide. The RT-PCR product of β-actin transcripts was used as an internal control. D, ITC-induced tubulin degradation is independent of protein translation and does not require de novo synthesis. HeLa cells were treated with 10 μg/ml cycloheximide (CHX) and/or 10 μm BITC for 8 h. The whole cell lysate was extracted and blotted for α-tubulin.