Mitochondrial Protein Extraction and Analysis

Purified mitochondria were incubated with protease K (for 25 min at 0°C) to remove the cytosolic proteins loosely associated with the outer mitochondrial membrane. The purified mitochondrial pellet was lysed in a buffer containing 50 mmol/L HEPES, 100 mmol/L NaCl, 1% NP-40, and a mixture of protease inhibitors (Roche Molecular Biochemicals, Mannheim Germany) and phosphatase inhibitors (Sigma). After homogenizing with 20 strokes using a Dounce homogenizer ( Bellco Glass, Vineland, NJ, U.S.A.), mitochondrial lysates were centrifuged at 20,000 g, and the supernatants were used for western blot analysis with anti-PGC-1α,25 (link) anti-SIRT1 (sc-15404; Santa Cruz Biotechnology, Dallas, Texas, U.S.A.), anti-TFAM (NBP1-71648; Novus Biologicals, Littleton, CO, U.S.A.), anti-pTau Ser404 (sc-12952; Santa Cruz Biotechnology), anti-pTau Ser396 (44752; Invitrogen), anti-pTau Ser262 (ab131354; AbCam, Cambridge, MA. U.S.A.), anti-pTau Thr 231 (AB 9668; Millipore, Billerica, MA, U.S.A.), anti-Histone H3 (ab1791; AbCam), anti-GAPDH (2118; Cell Signaling), or anti-HSP 60 (4870; Cell Signaling) antibodies. Horseradish peroxidase-conjugated secondary antibodies were purchased from Pierce Biotechnology, Rockford, IL, U.S.A. Antibody binding was detected by using the SuperSignal chemiluminescence kit (Pierce) and an Alpha, Innotech imaging system (Santa Clara, California, U.S.A).

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Choi J., Chandrasekaran K., Demarest T.G., Kristian T., Xu S., Vijaykumar K., Dsouza K.G., Qi N.R., Yarowsky P.J., Gallipoli R., Koch L.G., Fiskum G.M., Britton S.L, & Russell J.W. (2014). Brain diabetic neurodegeneration segregates with low intrinsic aerobic capacity. Annals of Clinical and Translational Neurology, 1(8), 589-604.

Publication 2014

protease inhibitors phosphatase inhibitors anti-pTau Thr 231 anti-Histone H3 anti-GAPDH (2118; anti-HSP 60 Horseradish peroxidase-conjugated secondary antibodies

Antibodies Antibody Biologicals Buffer Chemiluminescence Cytosolic Gapdh Hepes Histone h3 Horseradish peroxidase Inhibitors Mitochondrial Nacl Novus Np 40 Outer mitochondrial membrane Pgc 1α Phosphatase Protease Protease inhibitors Proteins Sirt1 Strokes Tfam Western blot analysis

Corresponding Organization : Veterans Health Administration

Other organizations : University of Michigan–Ann Arbor

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Variable analysis

independent variables

Incubation of purified mitochondria with protease K (for 25 min at 0°C)

dependent variables

Protein levels of PGC-1α, SIRT1, TFAM, pTau Ser404, pTau Ser396, pTau Ser262, pTau Thr 231 (measured by western blot analysis)

control variables

Mitochondrial lysates were prepared in a buffer containing 50 mmol/L HEPES, 100 mmol/L NaCl, 1% NP-40, and a mixture of protease and phosphatase inhibitors
Mitochondrial lysates were centrifuged at 20,000 g, and the supernatants were used for western blot analysis

controls

Positive controls: Anti-Histone H3, anti-GAPDH, and anti-HSP 60 antibodies were used as loading controls
Negative controls: Not explicitly mentioned

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