Figure EV5. cBID is targeted to damaged mitochondrial membranes (relates to Fig 6).

• A
  Mitochondria‐enriched membrane fraction from Bax ^−/− Bak ^−/− MEFs expressing BAK∆Cys were incubated with Ub^G76C (100 μM) in the presence or absence of oxidant copper phenanthroline (CuPhe, 1 mM) for 30 min prior to separation of supernatant and membrane fractions and immunoblotting as indicated. Note the incorporation of exogenous Ub^G76C into high molecular weight species (*) in the membrane fraction following treatment with oxidant. (**), Ub^G76C disulphide‐linked dimer.
• B
  HeLa cells expressing HA‐Parkin were treated with antimycin and oligomycin (AO) for 2 h prior to fractionation into cytosol (C) and heavy membrane (M). Membrane fractions were then incubated with cBID as indicated. Incorporated cBID was then determined by centrifugation to separate the supernatant (S) from the membrane pellet (P) prior to immunoblotting.
• C
  HeLa + HA‐Parkin cells were treated with antimycin and oligomycin (AO) for 2 h prior to harvesting. Samples were immunoblotted as described. Ubiquitinated MCL1 (arrowhead).

Source data are available online for this figure.