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. 2012 Aug 22;287(41):34635–34645. doi: 10.1074/jbc.M112.357509

FIGURE 2.

FIGURE 2.

ΔΨm loss induces the dynamic changes in the band pattern of PGAM5. A, mitochondria-damaging agents induce changes in the band pattern of PGAM5. HEK293A cells treated with 10 μMCCCP, 20 μg/ml ethidium bromide (EtBr), or 2 mm paraquat for indicated time periods were subjected to IB. B, mitochondrial fragmentation is dispensable for the changes in the band pattern of PGAM5. 48 h after co-transfection of Drp1 K38A-GFP and PGAM5-HA in HEK293A cells, cells were untreated or treated with 30 μm CCCP for 3 h and subjected to IB (upper panels) or immunocytochemistry (lower panels). Scale bar = 10 μm. C, mitochondrial fragmentation is not sufficient to change the band pattern of PGAM5. 24 h after co-transfection of FLAG-Fis1 and PGAM5-HA in HEK293A cells, cells were untreated or treated with 30 μm CCCP for 3 h and subjected to IB (upper panels) or immunocytochemistry (lower panels). Scale bar = 10 μm. D–F, the changes in the band pattern of PGAM5 are induced by the ΔΨm loss-inducing agents. HeLa cells were treated with 20 μm CCCP, 20 μm nigericin, 20 μm valinomycin, or 10 mm 2-deoxy-d-glucose (2DG)/10 μm oligomycin for the indicated time periods. The treatment with 2-deoxy-d-glucose/oligomycin was performed in glucose-free medium. After each stimulus, cells were lysed and subjected to IB (D). ΔΨm in D was detected with the ΔΨm-dependent dye, MitoTracker red CMXRos. Scale bar = 10 μm (E). Cellular ATP levels in D were quantified using the ATP detection kit (F).