Western blot analysis showed Beclin 1 cleavage products (<49 kDa) were not observed in primary neurons exposed to mitophagy-inducing sublethal rotenone (250 nM × 2h: Rot low) or staurosporine (100 nM × 2h: STS) concentrations (a). However, Beclin-1 was cleaved upon exposure to a lethal dose of rotenone (1 mM × 24h: Rot high). Likewise, SH-SY5Y cells did not show Beclin 1 cleavage upon exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin. Cell lysates from stable PINK1-Flag expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), Rot (1 μM), or FCCP (2 μM), and analyzed by Flag immunoblot for PINK1-Flag levels (d). SH-SY5Y cells transfected with HA-Parkin were treated the indicated toxins, immunolabeled for HA (green) and mitochondrial p60 antigen (red) and analyzed for HA-Parkin re-distribution to mitochondria (e). Scale bar: 10μm. FCCP elicited translocation of Parkin to mitochondria (e, arrows), which was not seen with the other mitophagy stimuli. Cytosolic and mitochondrial pellets were prepared from SHSY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or Rot (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers (f). Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or Rot treated cells. See for uncropped blots.