Cytochrome c release assay and western blot

Cells were seeded on a 12-well plate. For hydrogen peroxide treatment, reagent was added directly into the well to achieve the appropriate concentration. Separation of mitochondrial and cytosolic fractions were performed using the mitochondrial isolation kit from ThermoFisher (cat:89874) with an additional step of trichloroactic acid precipitation of the cytosolic fraction. The final pellet was dried in a 95°C heat block for 2–3 min before resuspending it in the SDS loading buffer. Cell lysates were separated by SDS-PAGE, transferred onto the nitrocellulose membrane and blocked in 5% milk in PBS containing 0.1% Tween. Primary and secondary antibodies were diluted in the blocking buffer and incubated at room temperature for 2 hr. The bands were detected using the electrochemiluminescence reagent and exposure onto x-ray films. The following antibodies were used in western blot: anti-mouse cytochrome c (Abcam: ab13575), anti-rabbit gamma tubulin (Sigma-Aldrich: T6557), anti-rabbit Rabenosyn-5/ZFYVE20, anti-rabbit Alsin (Sigma Aldrich: SAB4200137), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse APPL2 (home-made), anti-mouse GAPDH (Sigma Aldrich: G8795), anti-mouse gamma tubulin (Sigma Aldrich: T6557), and anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415).