FIGURE 4.

Inhibition of the E1 enzyme does not affect nucleolar stress-induced PICT1 degradation in ts85 cells. ts85 cells were incubated at either 33 or 39 °C for 18 h. Cell lysates were prepared, and ubiquitination (Ub) of cellular proteins was analyzed (A). After incubation at the indicated temperature, cells (3.5 × 10⁵ cells per 0.9 ml) were further incubated with 5 nm ActD (B) or 50 μg/ml cycloheximide (C) for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis as described under “Experimental Procedures.”