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. Author manuscript; available in PMC: 2014 Dec 19.
Published in final edited form as: J Biol Chem. 2005 Nov 22;281(4):2061–2070. doi: 10.1074/jbc.M507741200

Figure 1. Inhibition of CcO activity in macrophages during hypoxia and reversal by PKA inhibitors H89 and MPI.

Figure 1

RAW 264.7 mouse monocyte macrophages were grown under normoxia or hypoxia (0.1% O2 for 10 h) with or without added H89 or MPI (50 nm each) or 10 nm Go6850 and used for assays. A, CcO activity was measured in permeabilized macrophages grown under normoxia or hypoxia using the DAB method. B, CcO activity was measured spectrophotometrically following the oxidation of cytochrome c in enzyme reconstituted in asolectin-cardiolipin vesicles as described under “Materials and Methods.” C, PKA activity was measured in mitochondria and cytosol from macrophages grown under different conditions. D and E, CcO enzyme from cells grown under different conditions was solubilized in 1.5% sodium cholate and immunoprecipitated with the holoenzyme antibody. Equal amounts of immunoprecipitates were resolved on two companion gels, and the proteins were probed with polyclonal antibody against CcO (D) and antibody to Ser phosphate (E). The conditions for isolation of mitochondria, immunoprecipitation, and immunoblotting were as described under “Materials and Methods.” A–C, the average ± S.D. were calculated from 3 to 4 separate assays. Asterisks indicate significant differences (**, p =<0.01).