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Figure 3

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Effects of antioxidants on mitochondrial PKAα subunit level and activity.

RAW 264.7 macrophages were subjected to hypoxia for 12h with or without addition of 1µM Mito-CP or 10mM N-Acetyl Cysteine. At the end of hypoxia, part of the cells was used for measuring ROS production by DCFDA oxidation and the remaining for mitochondria isolation. Protein was estimated by Lowry’s method. A) PKA activity and ROS production (n=3). After hypoxia cells were plated in 96 well plate in phosphate buffered saline and incubated with DCFDA (1µM) for 15 minutes. Fluorescence was measured at Excitation 525nm and Emission 575nm. Corresponding PKA activity was measured in 10µg of mitochondrial protein, B) PKAα protein level. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. PKAα and CcO IVi1 antibodies were used for immunoblotting. Relative band intensities are given in parantheses. The blot is representative of two separate experiments. C) Effect of Mito-CP on CcO activity under hypoxia. CcO activity was measured with 10µg of mitochondria (n=4). **, p<0.005.

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