**3.3. Mitochondria and endoplasmic reticulum isolation from mouse hair cell culture**

The mitochondria were isolated from mouse cochlear hair cell cultures using a kit per manufacturer's instructions (Qproteome TM Qiagen). The cells were washed with PBS buffer and harvest these cells with 1 ml of disruption buffer containing protease inhibitor cocktail and incubated 10 min at RT. After 10 min the cells were centrifuged at 6000×g for 10 min and collect the pellet and discard the supernatant. The pellet was resuspended in purification buffer followed by spun at 20,800×g for 15 min. Mitochondria and ER were layered on the surface of a density gradient centrifugation. Both mitochondria and endoplasmic reticulum were removed from the respective gradient and diluted in storage buffer, and spun at 8000×g for 10 min. The pellet consisting of purified mitochondria and endoplasmic reticulum were either resuspended in storage buffer and store in −80C or resuspended in protein lysis buffer get mitochondria and endoplasmic reticulum proteins.
