2.9. Western blotting

Cells and aortas were lysed in RIPA buffer (Beyotime, Guangzhou, China), and 40 µg total protein was resolved in 12% or 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane, which was then blocked with 5% skimmed milk. Primary antibodies against iNOS (1:2,000, AF0199, Affinity, USA), COX-2 (1:2,000, AF7003, Affinity), CHOP (1:1,000, 2895, CST, USA), BCL-2 (1:1,000, YT0470, ImmunoWay, USA), BAX (1:1,000, YT0455, ImmunoWay), cleaved Caspase 3 (1:1,000, AF7022, Affinity), cleaved Caspase 8 (1:1,000, AF5267, Affinity), TIPE2 (1:1,000, DF3326, Affinity), TLR4 (1:2,000, AF7017, Affinity), TRIF (1:2,000, DF6289, Affinity), MyD88 (1:2,000, AF5195, Affinity), AKT (1:1000, YM3618, ImmunoWay), p-AKT^Ser473 (1:1000, YP0006, ImmunoWay), and β-actin (1:10,000, AF7018, Affinity) were used. HRP-conjugated corresponding secondary antibodies (1:10,000, S0001 and S0002, Affinity) were used, and antigen–antibody reactions were visualized using an enhanced chemiluminescence detection kit (E411-05, Vazyme, China). ImageJ 1.48 (NIH, Maryland, USA) was used to quantify the intensities of each band. β-actin served as an internal reference.