Fig 5.

(A to E) Effect of wild-type NPM or NPM mutant expression on stress-induced mitochondrial NPM and Bax accumulation and outer mitochondrial membrane injury. (A) Immunoblot analysis of NPM (upper) and Bax (lower) accumulation in the mitochondrial fraction of cells expressing either empty vector (EV), NPM lacking the nuclear localizing sequence (NPM-ΔNLS), or wild-type NPM (NPM-WT) at baseline (Base), after 60 min of ATP depletion (ATP), or after 20 min of recovery (Rec). F₁F₀-ATPase, an intrinsic outer membrane protein, was used as the loading control. (B) Nonmitochondrial AIF and cytochrome c (Cyto-C) in cells before and after stress (assessed in the presence of high-dose digitonin; see Materials and Methods), with GAPDH as a loading control. (C) Densitometric analysis of 3 to 4 separate immunoblot studies performed as described for panel B. (D) Immunoblot analysis of active caspase 3 (17- and 19-kDa bands) in lysates harvested from cells that express either empty vector (EV), NPM lacking the nuclear localizing sequence (NPM-ΔNLS), or wild-type NPM (NPM-WT) at baseline (Base), immediately after 60 min of ATP depletion (ATP), and after 60 min of recovery (Rec). (E) Densitometric analysis of 3 to 4 separate immunoblot studies performed as described for panel D.