Figure 3.
PHB is present in channel-forming fraction and is increased upon activation of mPTP. (a) Thin-layer chromatography (TLC) showing a mix of phospholipid standards (lane 1, CL: cardiolipin; PE phosphatidylethanoamine; PI, phosphatidylinositol; PS, phosphatidylserine) and synthetic PHB standard (lane 2). (b) TLC showing mitochondrial extract (lane 1) and a PHB standard (lane 2). Both TLCs were stained with Iodine. (c) PHB detection using dot blot immunostaining of the extracts using PHB antibodies. Note that the signal was reduced by PHB-depolymerase (PhaZ7) treatment. PHB was present in higher amounts in calcium-treated mitochondria -Ca2+(mPTP)- compared with control or mitochondria pretreated with Cyclosporin A (+CSA) or Ruthenium Red (+RR). (d) Densitometry analysis of dot blots from four independent experiments showing a significant increase in the recovery of PHB in the extract from mitochondria treated with calcium when compared with control, RR- or CSA-treated mitochondria (n=4, *P<0.05, t-test).