Hrp conjugated anti rabbit immunoglobulin g antibody

After rinsing in cold PBS three times, the cells were homogenized in RIPA buffer. The supernatant was then centrifuged at 120,000×g for 15 min at 4 °C. The samples (10–20 mg) were run on SDS-PAGE gels, transferred to PVDF filter membranes, and used for western blotting with monoclonal antibodies against CaMKIIδ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p47^phox, Nox2/gp91^phox (Abcam, Cambridge, MA, USA), cleaved caspase-3 (Asp175) (Cell Signaling Technology), and ApoJ (EterLife, Birmingham, UK). The PVDF membranes were then incubated with HRP-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The blot was developed with an ECL-Plus chemiluminescence reagent kit and visualized with the UVP Bio-Imaging System. The blot densities were analyzed using Image J.

After being rinsed in cold PBS three times, cells were homogenized in RIPA buffer. The supernatant was then centrifuged at 120,000 × g for 15 min at 4 °C. Samples (10–20 mg) were run on SDS-PAGE gels, transferred to PVDF filter membranes and Western blotted with monoclonal antibodies against phosphor-Akt (Ser473) (Cell Signaling Technology, Beverly, MA, USA), Nox2/gp91^phox (Abcam, Cambridge, MA, USA), Phospho-NF-κB p65 (Ser536) (Cell Signaling Technology), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), cleaved caspase-3 (Asp175) (Cell Signaling Technology), ApoJ (EterLife, Birmingham, UK) and β-Actin (Sigma). PVDF membranes were then incubated with HRP-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The blot was developed with an ECL-Plus chemiluminescence reagent kit and visualized with the UVP Bio-Imaging System. Blot densities were analyzed using Image J software.