Camkiiδ

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.

Western blot test was used to identify expression of SMC proteins. Cells were lysed in RIPA buffer with protease and phenylmethylsulfonyl fluoride (PMSF; Roche, Indianapolis, IN, USA). The protein concentration was assayed using the BCA method (Bio-Rad). Approximately 20 to 50 μg of total protein samples was loaded on a 10% SDS-PAGE after denaturation by boiling for 10 min. The separated proteins transferred to polyvinylidene difluoride membranes. Membranes were blocked by incubation in Tris-buffered saline containing 0.05% Tween 20 and 5% skimmed milk with constant shaking for 1 h. The membrane was probed with antibodies against a-SMA (1 : 1000, Abcam, Cambridge, UK), calponin (1 : 1000, Abcam, Cambridge, UK), SM22a (1 : 1000, Abcam, Cambridge, UK), SM-MHC (1 : 1000, Abcam, Cambridge, UK), smoothelin-like 2 (1 : 2000, Santa Cruz, Dallas, USA), ACLP (1 : 1000, Abcam, Cambridge, UK), CaMKIIα (1 : 5000, Abcam, Cambridge, UK), CaMKIIβ (1 : 1000, Abcam, Cambridge, UK), CaMKIIγ (1 : 500, Abcam, Cambridge, UK), and CaMKIIδ (1 : 1000, Santa Cruz, Dallas, USA) overnight at 4°C. GAPDH was used as an internal loading control. The membranes were washed three times with TBST and incubated with the appropriate secondary antibodies at room temperature for 1 h and detected using enhanced chemiluminescence.