FIG 6.

Inhibition of mTOR signaling reduces 28S maturation. (A, upper) The localization of SENP3 and NPM1 was monitored by immunofluorescence in HeLa cells after the siRNA-mediated depletion of Rictor or Raptor. (Lower) Efficient depletion of the respective proteins was verified by immunoblotting with anti-Raptor or anti-Rictor antibody with antitubulin serving as a loading control. (B) A pulse-chase rRNA processing assay, as described in the legend to Fig. 1D, was performed in HeLa cells after siRNA-mediated depletion of SENP3 or treatment of cells with Ku-0063794. After extraction, the RNA was run on a denaturing agarose gel, stained with EtBr, and subjected to autoradiography for the detection of the various rRNA species. The efficiency of depletion and levels of SENP3 was controlled by Western blotting with anti-SENP3 antibody. Vinculin was used as a loading control. The 28/32S rRNA ratio was quantified using a phosphorimager. The values represent averages from 3 independent experiments, and the error bars indicate standard errors of the means. (C) As described for panel B, except the cells first were transfected with control siRNA or siRNA directed against SENP3 or Raptor before the pulse-chase assay. The efficient depletion of the respective proteins was monitored by immunoblotting with anti-SENP3 and anti-Raptor antibodies, using tubulin as a loading control. Phosphorimager quantifications are as shown in the lower panel.