FIG. 1.

Mitochondrial localization of PNPase. (A) Recombinant human PNPase expression and detection. Immunoblots of whole-cell lysates from HEK293T cells mock (M) transfected or transfected with a 5′-myc-PNPase (5′m) expression construct are shown. (B) Exogenous PNPase localizes to mitochondria. HEK293T cells were mock (M) or empty vector (V) transfected or transfected with a PNPase-3′-myc (3′m) expression construct, and whole-cell lysates (W) and cytosolic (C) and mitochondrial (MT) fractions (Fxn) were probed for the myc epitope tag (α-m), for cytochrome c (α-c), or for PNPase (α-PNP). Asterisks indicate PNPase; additional bands are nonspecific. (C) Preexisting and IFN-β-induced PNPase localizes to P3HR-1 Burkitt lymphoma cell mitochondria. Cells were left untreated or were incubated with 1,000 U/ml IFN-β for 24 h, and whole-cell lysates (W) and cytosolic (C) and mitochondrial (MT) fractions were probed for PNPase and β-actin. (D) PNPase immunolocalizes to the mitochondria. MeWo melanoma cells were incubated with MitoTracker Green FM dye and PNPase peptide-purified rabbit antibody, followed by PE-conjugated anti-rabbit IgG. The right panel shows the images merged.