Figure 5.

Phosphomimic nucleophosmin (NPM) proteins that replicate stress-induced phosphorylation changes regulate NPM toxicity. The biologic behavior of nucleophosmin (NPM) phosphomimic proteins assessed using functional bioassays performed in primary murine proximal tubule epithelial cells (PTECs) that express flag-tagged wild type (WT), a normal (Nor-M or NrNPM) NPM mimic protein, or a stress (Stre-M, StNPM, or Stress-M) NPM mimic protein that replicates differential NPM phosphorylation. (A) NPM localization in intact renal cells in the absence of stress. (B) NPM oligomers and monomers detected in lysates harvested from nonstressed cells in a native gel; total NPM content is unchanged in each lane, and the increase in monomeric NPM is accompanied by a reciprocal decrease in the oligomeric form of NPM. (C) NPM-Bax complex formation assessed after immunoprecipitation (IP) of flag-tagged NPM followed by immunoblot (IB) of conformationally activated Bax (Bax 6A7) after ischemic stress; input protein amounts were similar in each sample. (D) Accumulation of NPM and 6A7 Bax in isolated PTEC mitochondria after ischemic stress; VDAC, a mitochondrial membrane protein, serves as loading control. (E) Cell survival after 60 minutes ATP depletion (n=6). EV, empty sgOpti vector; RDU, relative density unit. *P<0.05 for Nor-M and Stress-M NPM phosphomimics versus control (CTL).