Figure 1.

Stress causes cytosolic nucleophosmin (NPM) translocation. (A) Quantitative assessment of NPM in the nuclear and cytosolic cell fractions of primary human proximal tubule epithelial cells (PTECs) subjected to 60 minutes ATP depletion; identical amounts of proteins were loaded in each lane, and densitometric analysis was used to assess the relative amount of NPM present in the cytosolic and noncytosolic fractions before (Base) and after ATP depletion (ATP Depl; n=3). NPM accumulation detected in the cytosolic fraction of (B) renal cortical homogenates harvested from paired donor kidneys with either normal perfusion (normal) or perfusion pump failure (ischemic; results represent four kidneys from two human donors), (C) primary murine or human PTECs subjected to ATP depletion ×60 minutes, and (D) murine and human PTECs subjected to 70 minutes of hypoxia. In B–D, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a loading control; densitometry represents three independent experiments in each study. Cytosolic fractions were harvested using digitonin (see Methods); noncytosolic extracts were harvested by exposing cells to RIPA buffer after extracting the cytosolic fraction. CTL, control; RDU, relative density unit.