FIGURE 2.

Ras/NORE1A stabilizes PP1A. A, HEK-293 cells were transfected with PP1A, NORE1A, and activated H-Ras expression constructs for 24 h. The cells were treated with cycloheximide (20 μg/ml) and lysed at the indicated times after addition of cycloheximide. Levels of PP1A protein were measured by Western blotting analysis. Shown is a representative blot of three independent experiments. IB, immunoblot. B, the density of the bands was quantitated using ImageJ software, and relative PP1A expression was calculated after normalizing to β-Actin expression. No significant difference in PP1A expression levels was found in cells transfected with NORE1A or Ras individually. However, in cells transfected with both NORE1A and Ras, there was a significant increase in the levels of PP1A. C, HBEC-3KT cells stably knocked down for NORE1A were induced to express activated Ras. 3 days after induction, cell lysates were prepared and analyzed for PP1A expression by Western blotting analysis.