Fig. 4. Effect of paclitaxel on steady state tubulin accumulation. Cells were incubated overnight (16-24 hours) in the presence of [³H]methionine and the indicated concentrations of paclitaxel to measure the amount of protein that accumulated. Following lysis in SDS, the cellular contents were mixed with a constant volume of [³⁵S]methionine-labeled wild-type CHO extract, precipitated with acetone, and resolubilized in urea sample buffer for 2D gel analysis. Spots representing ß-tubulin and actin were excised from the gels, solubilized, and analyzed by liquid scintillation counting to determine their 3H/35S ratios. Each isotope ratio for tubulin was normalized by dividing by the isotope ratio for actin in the same sample, and the resulting values were expressed relative to untreated wild-type (WT) cells set at 100% (A) or relative to the zero concentration control for each cell line (B). The values represent the mean from 3-8 independent experiments. Representative standard deviations are shown only for WT and Tax 2-4. The inset in B shows an enlargement of the lower paclitaxel concentrations for WT, CV 2-8 and Tax 5-6.