![Figure 5. Actinonin impairs the turnover of de novo mitochondrial protein synthesis. (A, left) Standard protocol for metabolic labeling of HEK293 cells after ATP5B (A-5B) siRNA knockdown or scrambled control (SC) with a chase. Arrows indicate the position of MT-ATP6 and MT-ATP8. (right) SDS-PAGE immunoblot of whole cell lysates with representative antibodies. (B) Modified protocol for extended metabolic labeling of HEK293 cells after ATP5B siRNA knockdown for a 3-h pulse and chase in the indicated conditions (Act, actinonin; E, ethanol). (C) Immunoblot of whole cell lysates of ATP5B siRNA knockdown treated with actinonin or ethanol for 6 h. (D) HEK293 cells metabolically labeled for 45 min with [35S]methionine/cysteine with and without actinonin and then precipitated with CTAB. Each sample was split equally in two with one half treated with RNase A before addition of CTAB. A representative image of three independent experiments is shown.](https://cdn.rupress.org/rup/content_public/journal/jcb/211/2/10.1083_jcb.201504062/6/jcb_201504062_fig5.jpeg?Expires=1767783526&Signature=sjZUIF4iDhnJ8KiKnCvcsAbA4a8eLowIYidFjWJH9fHWNqhv5ulEfi16ALtMZQ8452ap0UHAzq3fTxtfqedL--BnHp8G2kTqRcPqTUpsnvAud1sWEEK4slZBrnn7Z629mQsgvEVDOWwzFc5t02q8itO1rX3iDHgIplKkH3oQE0r~h~nILy~Ljc8twUYAiFz-U53tNo9A75pruu-Cx9P7CWCOoRJp-y6hndU9NbCIYYpuq~BUbRJ8R~lb2bGXpwPbNG2kW3s-N8WylVhA9tXiWvlaWyboif~vlXVaNLsyd6NTnHcuGio2OcLVEdFgXe9K7NBQ2Egi9y7iK52a70sHwQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Actinonin impairs the turnover of de novo mitochondrial protein synthesis. (A, left) Standard protocol for metabolic labeling of HEK293 cells after ATP5B (A-5B) siRNA knockdown or scrambled control (SC) with a chase. Arrows indicate the position of MT-ATP6 and MT-ATP8. (right) SDS-PAGE immunoblot of whole cell lysates with representative antibodies. (B) Modified protocol for extended metabolic labeling of HEK293 cells after ATP5B siRNA knockdown for a 3-h pulse and chase in the indicated conditions (Act, actinonin; E, ethanol). (C) Immunoblot of whole cell lysates of ATP5B siRNA knockdown treated with actinonin or ethanol for 6 h. (D) HEK293 cells metabolically labeled for 45 min with [35S]methionine/cysteine with and without actinonin and then precipitated with CTAB. Each sample was split equally in two with one half treated with RNase A before addition of CTAB. A representative image of three independent experiments is shown.
