Figure 4.

Nucleophosmin protein expression and rate of nucleophosmin mRNA translation is higher in MEFs lacking Tsc1. A, equal amounts of protein lysate from WT, Tsc1^−/−/p53^−/−, or p53^−/−MEFs were resolved by SDS-PAGE and analyzed with antibodies specific for TSC1, γ-tubulin, and nucleophosmin. B, Tsc1^−/−/p53^−/− MEFs were treated with vehicle or rapamycin (100 nmol/L) and harvested 24 h later for Western blot analysis. Proteins were resolved by SDS-PAGE and antibodies recognizing γ-tubulin, nucleophosmin, and phospho-S6 were used. WT and Tsc1^−/−/p53^−/− MEFs were transduced with plasmids encoding β-galactosidase (+ Vector) or TSC1 and harvested 24 h later for Western blot analysis. Proteins were resolved by SDS-PAGE and antibodies recognizing γ-tubulin, nucleophosmin, and TSC1 were used. C, WT MEFs were transduced with pRK7 plasmids encoding HA-tagged S6 kinase (S6K) 1 or eIF4E. Cells were harvested (48 h later) and proteins were separated by SDS-PAGE and immunoblotted with antibodies specific for HA, γ-tubulin, and nucleophosmin. D, WT, Tsc1^−/−/p53^−/−, or p53^−/− MEFs were starved of methionine, and [³⁵S]methionine was subsequently added for the indicated times before harvesting to allow for incorporation of label into newly translated proteins in the absence or presence of rapamycin (100 nmol/L). Immunoprecipitated (IP) nucleophosmin proteins were separated by SDS-PAGE and detected by autoradiography.