Western blot and quantitation analysis of protein expression and ATP levels.
Homogenates and detergent solubilized extracts of tissues and immunoblots were prepared as described elsewhere [69]. Quantitation of immunoblot bands calibrated against internal marker bands, such as mHsp70 and Nup153, was carried out with Gel Pro Analyzer (MediaCybernetics, San Diego, California, United States). For determination of ATP levels, freshly dissected and flash-frozen tissues were homogenized in 2.5% trichloroacetic acid (TCA), neutralized, and diluted to a final concentration of 0.1% with Tris-Acetate (pH 7.75). ATP measurements were carried out with the ENLITEN ATP Assay System Bioluminescence Detection Kit for ATP (Promega, Madison, Wisconsin, United States) as per the manufacturer's instruction in SpectraMax-M5 (Molecular Devices, Sunnyvale, California, United States). Protein concentration was measured by the BCA method (BioRad, Hercules, California, United States) using BSA as a standard.