Rapamycin reverses effects of parkin activity loss 1015 Figure 3. Immunocytochemistry (ICC) analysis of mitochondrial localization of parkin in various conditions. Z-stack images of parkin (green), mitotracker red (red), DAPI (blue), and merged (yellow) were captured using LSM510 confocal microscopy, white arrows indicating formation of yellow punctae. (A) MAO-B, (B) MAO-B, (C) MAO-B, 1 μM rapamycin pre-treatment, (D) MAO-B, 4 μM FCCP treatment, (E) MAO-B, 4 μM FCCP treatment, (F) MAO-B, FCCP, rapamycin, (G) MAO-B, FCCP, rapamycin. capable of tagging damaged mitochondria for lyso- somal removal due to corresponding losses in parkin ubiquitin E3 ligase activity. We examined the effects of MAO-B induction on mitophagy and whether this process was impacted by pre-treatment with rapamy- cin. To perform these studies, parkin-MAO-B cells were ﬁxed and analysed for alterations in the autophagic marker LC3 and parkin expression in the various cellular conditions. Our experiments demon- strated a decrease in LC3 following induction of human MAO-B expression, suggesting decreased efﬁciency of autophagic degradation (Figure 5). This Figure 4. Cellular parkin levels in various conditions.Whole cell lysates fractions were analysed via Western blot analysis using ﬂag antibody (Sigma); β-actin was used as loading control (Chemicon). Corresponding densitometric analyses are shown (n 3). Values are expressed as mean SD; ∗∗∗p 0.001, ∗∗p 0.01 versus MAO-B cells.