Figure 4.

Treatment of cells with LMB and CHX results in a nuclear accumulation of c-Abl. wt c-Abl expressing cells (panels A–F) were pretreated with CHX for 30 min and then further incubated with (D–F) or without (A–C) 5 nM LMB for 6 hr. Cells were then fixed and stained with the anti-Abl 8E9 antibody (A, D), Hoechst dye (DNA, B and E) and phalloidin (actin). The merged images of the 8E9 and phalloidin staining are shown in C and F. Comparison of D and A or F and C shows that cytoplasmic c-Abl is lost in LMB-treated cells. To test whether CHX+LMB treatment caused a nonspecific loss of cytoplasmic c-Abl, c-Abl NLS(−) expressing cells were similarly treated with 5 nM LMB for 6 hr, either in the presence (G and H) or absence (I and J) of CHX. The anti-Abl stain (G and I) and the Hoechst stain (H and J) were shown. The intensity of Abl stain was not diminished after CHX-LMB treatment, showing that the cytoplasmic pool of Abl is stable under the experimental conditions.