Ab150422

Proteins were extracted from tissue homogenates. For Western blot analysis, protein samples were fractionated in 4–20% SurePAGE™ Precast Gels (M00657, GenScript Biotech Corporation, China) and transferred to nitrocellulose membranes. The analysis was performed with anti-CCN1 (ab228592, Abcam, UK), anti-CDC42 (2466T, Cell Signaling Technology, USA), anti-Claudin5 (4C3C2, Invitrogen, USA), anti-COX6c (ab150422, Abcam, UK), anti-CREB1 (R23983, ZEN- BIOSCIENCE, China), anti-HIF1α (ab179483, Abcam, UK), anti-NDUFα1 (15561-1-AP, Proteintech Group, USA), anti- SEPN1 (55333-1-AP, Proteintech Group, USA), anti-SHP1 (ab32559, Abcam, UK, anti-VEGFa (NB100-664, Novus Biologicals, USA), and anti-β-tubulin (2146S, Cell Signaling Technology, USA) antibodies. Immunoassays were performed using enhanced chemiluminescence (17046; ZEN-BIOSCIENCE, China), in accordance with the manufacturer’s instructions. Protein levels were determined by densitometric scanning of protein bands. Six retinas were used in each group, and the results for each retina were averaged from three replicates.

The mitochondrial protein sample was mixed with the loading buffer and heated at 100 ℃ for 8 min, then separated by SDS-PAGE (Tris-HEPES-SDS gradient 4 to 20% gels) and transferred to a PVDF membrane. The membrane containing the transferred protein was blocked with 5% skim milk (150 mM NaCl, 10 mM Tris, 0.1% Tween-20, pH 8.0) in TBST. The blocked membrane was incubated with anti-β-actin (1:1000, Santa Cruz, CA, sc-47778), anti-histone 3 (1:1000, Abcam, ab1791), anti-VDAC1 (1:2000, Abcam, ab154856), anti-caspase-3 (1:1000, CST, 9665S), anti-cleaved caspase-3 (1:1000, CST, 9664L), anti-PURA (1:1000, Abcam, ab84928), anti-PSPC1 (1:1000, Abcam, ab184123), anti-Dynamin-1 (1:2000, Abcam, ab40758), anti-SYN2 (1:5000, Abcam, ab76494), anti-cytochrome c oxidase subunit (1:2000, Abcam, ab150422), anti-Complex I (1:1000, Abcam, ab110245), anti-beta tubulind (1:1000, Abcam, ab6046) in TBST buffer 4 ℃ overnight. After washing in TBST, membranes were incubated with a 1:3000 dilution of anti-rabbit or anti-mouse IgG HRP secondary antibody diluted in TBST for 1 h. Subsequently, the membrane was washed in TBST (4 × 10 min) and developed using a chemiluminescent reagent from an ECL kit (Thermo Scientific Pierce ECL, USA). Blots were detected on a phosphor imager and analyzed using ImageQuant 350 software.