Anti pkcε

Total proteins isolated from LV tissues were rapidly minced and homogenized in 1 × RIPA ice‐cold lysis buffer (with protease inhibitor). After centrifuging at 800 g for 5 min. at 4°C to remove nuclei, the supernatant was further centrifuged at 12,000 g for 30 min. to obtain the mitochondrial pellets and the cytosolic extracts (supernatant). Equal amount of mitochondrial fractions or cytosolic proteins was separated in 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore). The membranes were immunoblotted with anti‐Drp1Ser⁶¹⁶, anti‐caspase9, anti‐PKC‐δ, anti‐PKC‐ε, anti‐GSK‐3β and anti‐GSK‐3β^{Ser 9} (Cell Signaling, Beverly, MA, USA) at 4°C overnight. After washing by 0.1%PBS for three times, the blots were incubated with HRP‐conjugated anti‐IgG for 2 hrs. Immunoreactivities were detected using the enhanced chemiluminescence reaction system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Densitometric analysis was performed using QuantityOne software version 4.5.2 (Bio‐Rad, Hercules, CA, USA). In brief, the density area of each band can be automatically identified and outlined by the software, and then the brightness value for each band was obtained. The ratio of each detected protein to β‐actin represented to their relative protein levels.