Enhanced chemiluminescence reaction system

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.

After the exposure for 48 h, floating and adherent cells were harvested and rinsed twice with cold PBS and lysed in lysis buffer [50 mM Tris (pH 7.4), 400 mM NaCl, 50 mM NaF, 30 mM sodium PP_i, 1 mM sodium pyrovendidate, 1% SDS, and 0.5% NP40]. Protein concentrations were determined with bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Cellular proteins, at a concentration of 40 µg, were fractionated on 12% SDS-PAGE (Invitrogen/Novex, Carlsbad, CA, USA). Proteins were then transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA, USA). The membrane was blocked with 5% nonfat milk in TBST and incubated overnight with antibody at 4 °C. After washing three times with TBST, the membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody diluted with TBST (1:1000). The detected protein signals were visualized by an enhanced chemiluminescence reaction system (Amersham, Arlington Heights, IL, USA), and the signal was visualized with X-ray film (Hyperfilm; Amersham).

The cortex tissue of the injection area was collected for further analysis. All of the nuclear and cytoplasmic proteins (100 μg) of the brain tissue were size‐fractionated using SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to Immobilon‐P membranes (Millipore). The blotted membranes were incubated with primary antibodies against GABAR‐1/2, GAPDH (Abcam, HK Ltd), followed by incubation with an HRP‐conjugated secondary antibody (Jackson). The immunoreactivity was detected using an enhanced chemiluminescence reaction system (Amersham Pharmacia Biotech).