PMSF（苯甲基六甲酰氟）-BIOFOUNT

resorufin occurs in the mitochondrial matrix. (a) Confocal images of isolated liver mitochondria (average intensity projections of 10 Â 0.1 μM confocal planes captured at 1 Airy unit) showing resorufin and Mitotracker Green fluorescence. Resorufin fluorescence can be seen throughout the mitochondria matrix. Scale bar is 5 mM. (b) Mean line profile intensities of resorufin (RR) fluorescence through the central plane image of intact mitochondria after normalising both intensity (maxima ¼1) and size (edges and centre defined to set all objects to an equal, arbitrary diameter). N ¼ 13. Full width half maximum of the resorufin distribution is smaller than that of Mitotracker Green (MTG, p o 0.001, paired t test). (c) Western blots of mitochondrial subfraction marker proteins. AIF, apoptosis inducing factor; NDUFA9, NADH dehydrogenase 1 alpha subcomplex 9 (a component of complex I in the inner membrane); GDH, glutamate dehydrogenase. (d) Representative traces of resorufin fluorescence following incubation of the indicated liver mitochondrial subfraction with AR in the absence of HRP or substrate. All reactions are PMSF sensitive. (e) Resorufin fluorescence measured after incubation of intact liver mitochondria with AR for 10 min. Fluorescence (no PMSF, no further AR) was measured from intact mitochondria (ARþ mt) and after removing the outer membrane and intermembrane space (ARþ shaved mtp). Controls were mitochondria incubated without AR (mt) and blanks. Data are mean 7 SD, n¼ 3. (f) Representative traces of resorufin fluorescence in assay medium containing AR-pre-incubated liver (red circle) or brain (blue circle) mitochondria as only source of AR. Liver mitochondria were pre-treated (red diamonds) or not (red circle) with PMSF. 50mM AR without mitochondria was used as positive control. 49 pmole H 2 O 2 were added at the indicated time point. (g) Quantification of the experiment in f. Data are mean7 SD, n¼ 4. All values are significantly above 0 (Po 0.05, t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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