Figure 5.

PARK2 localization at MAM. (A-C) shSCR and shBECN1 SH-SY5Y cells, overexpressing YFP-PARK2, were treated with DMSO or 25 μM CCCP for 6 h. (A) Immunoblots of subcellular fractions subjected to gradient centrifugation, resolved in SDS-PAGE and revealed with specific antibodies. VDAC1 was used as a marker of pure mitochondria, whereas ACSL4 was adopted as a MAM-specific marker. PNS, post-nuclear supernatant; MitoP, pure mitochondria; MAM, mitochondria-associated membranes. PARK2 accumulation after CCCP treatment is more appreciable at higher exposure; the upper bands evident at lower exposure reflect PARK2 ubiquitination. Separated lines indicate that we joined together distant parts from the same gel. (B) Confocal analysis of YFP-PARK2 and the MAM-localized protein HSPA9. The insets display enlarged views of colocalized areas. Scale bar: 10 μm. (C) Histogram reporting Mander's overlap coefficients relative to YFP-PARK2 and HSPA9 colocalization shown in (B) (mean ± SD of n = 3, 10 cells per experiments). **p < 0.001.