Expression of PO in stably transfected U87 clones.

(A,B) Western blot analysis performed using mouse anti-GFP or rabbit anti-PO antibodies confirms the expression of both the fluorescent PO-EYFP wild-type and L441P fusion proteins in transfected U87 cells (A) or the untagged proteins (B). The same amount of sample, corresponding to 5 x 10⁴ cells or 50 μg of crude extract (EG), was loaded in each lane, as further confirmed by an anti-actin antibody. The level of endogenous PO in U87 control cells is below the detection limit (1 ng). The PO-EYFP L441P variant detected with the anti-PO antibody shows a further band at lower molecular mass corresponding to a partial proteolyzed form of the protein. (C, D) Confocal analysis of PO subcellular distribution in cultured U87 glioblastoma cells stably expressing PO-EYFP wild-type or PO-EYFP L441P. Both chimeric proteins display a perinuclear “spaghetti-like” distribution (yellow channel) which largely overlaps (merge panel) with the mitochondria-specific immunostaining signal (red channel). Western blot experiments were replicated four times. Scale bar: 5 μm.