Ab21058

The integrity and purity of the mitochondria fraction were confirmed by western blot analysis for several protein markers. Twenty micrograms of protein were separated by 4–12% gradient SDS-PAGE, transferred onto a PVDF membrane, electrotransferred to polyvinylidene difluoride membrane (PVDF) (Millipore, Billerica, MA), and probed with primary antibody followed by incubation with horseradish peroxidase-labeled IgG (1:5000) depending on the primary antibody. The purity of the mitochondria fraction was tested by screening for mitochondria protein utilizing the following antibodies: anti-VDAC (1:1000, 48661, cell signaling, CST), anti-β -tubulin (1:5000, ab21058, Abcam) for the cytosol, anti-calnexin for the nuclear and endoplasmic reticulum (2433 S, cell signaling, CST) in the isolated mitochondria versus in the supernatant fraction. We found that all mitochondria preparations were essentially free of cytosolic and nuclear contaminants. Together, these results attest to the purity of our mitochondria fractions, i.e., they were substantially free of cytosolic, nuclear or endoplasmic reticulum components.

Hippocampal neurons at 18–21 DIV were lysed after stimulation in lysis buffer that contained 20 mM Tris-Cl (pH 6.8) at 4 °C, 137 mM NaCl, 25 mM β-glycerophosphate, 2 mM NaPPi, 2 mM EDTA, 1 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 10 μl/ml protease inhibitor mixture (Sigma-Aldrich). Samples of culture media and lysates that contained equal amount of protein were mixed with 4× sodium dodecyl sulfate (SDS) sample buffer and denaturated by heating at 95 °C. The samples (20 μg) were subjected to 12% SDS-polyacrylamide gel electrophoresis and then electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). Ponceau S staining was performed to locate the protein bands on Western blots. The membranes were then blocked in 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated at 4 °C overnight with anti-β-dystroglycan (1:500; NCL-b-DG, Novocastra), anti-β-tubulin (1:1000; ab21058, Abcam), and anti-TIMP-1 (1:500; MAB580, R&D System) diluted in 10% nonfat milk in TBST. The membranes were then incubated with peroxidase-labeled secondary antibodies diluted 1:10000 in 10% nonfat milk in TBST for 1 h at room temperature. ECL Plus reagent (GE Healthcare) was used to detect horseradish peroxidase (HRP) on the immunoblots.