Protein Extraction from Mouse Cochleae

As in our prior experiments (Chen et al., 2012 (link); Zheng et al., 2014 (link); Yuan et al., 2015 (link)), we adapted our standard protocols as follows “Both inner ears were rapidly removed and dissected in ice-cold PBS, pH7.4, containing complete™ mini EDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich, Burlington, MA, USA, #11836170001) to remove vestibular portions. To extract total protein, tissues from two cochleae of a mouse were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich, R0278) with the cocktail proteinase inhibitors plus Phosphatase Inhibitor Cocktails II and III (Sigma-Aldrich, #P5726 and #P0044) by using a glass/glass micro–tissue grind pestle and vessel for 30 s. After 30 min on ice, tissue debris was removed by centrifugation at 15,000 × g at 4°C for 10 min and the supernatants were retained as the total protein fractions. Protein concentrations were determined using the Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA, #500-0114) with bovine serum albumin as a protein standard. Finally, the total protein was stored at −80°C after quantification.”