Df6251

Proteins from ischemic penumbral tissues and HAPI cells were used for Western blot analysis. SDS-PAGE was performed, and the resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by electroblotting. The membranes were blocked at Tris–buffered saline (TBST) containing 5% non-fat milk powder for 2 h and then incubated with the following primary antibodies overnight at 4°C: Arg1 (1:500, GTX109242, GeneTex), CD163 (1:500, WH112776, ABclonal), iNOS (1:500, GTX130246, GeneTex), CD86 (1:500, WH141312, ABclonal), P62 (1:200, WH146703, ABclonal), IRF5 (1:1000, ab181553, Abcam), TRAF6 (1:200, sc-8409, Santa Cruz), IKKαβ (1:200, BM4499, Boster), IL-10 (1:200, 20850-1-AP, Proteintech), IL-4 (1:500, 66142-1-lg, Proteintech), TNF-α (1:200, YT4689, ImmunoWay), and IL-1β (1:500, DF6251, Affinity). After being washed with TBST 3 times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse secondary antibodies for 2 h at RT. β-actin was used as a loading control. Immunoreactive bands were detected by an enhanced chemiluminescence (ECL) detection system. The gray values of the protein bands were quantified by Image Lab.

Prepared protein samples (10 μg) were subject to 12% SDS-PAGE, followed by electro-transfer onto PVDF membranes. Then the membranes were blocked in 5% skimmed milk for 1 h. Antibodies against exosome markers CD9 (ab92726; Abcam, Cambridge, UK), CD81 (ab109201; Abcam) and calnexin (ab133615; Abcam) were diluted at a ratio of 1:1000. The inflammatory response of vascular tissue and VSMCs was detected using antibodies against NFATc3 (18222-1-AP, 1:1000 dilution; Proteintech, Wuhan, China) and IL-6 (DF6087, 1:500 dilution; Affinity Biosciences, Cincinnati, USA), IL-1β (DF6251, 1:500 dilution; Affinity Biosciences) and TNF-α (ab205587, 1:500 dilution; Abcam). The internal control anti-β-actin primary antibody (TA-09; Zhongshan Jinqiao Biotechnology, Beijing, China) was used at 1:1000 dilution. The PVDF membranes were incubated with the above primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated goat anti-mouse IgG (ZB-2305, 1:4000; Zhongshan Jinqiao Biotechnology) or HRP-conjugated goat anti-rabbit IgG (ZB-2301, 1:4000; Zhongshan Jinqiao Biotechnology) secondary antibodies for 1 h at room temperature. After extensive wash, protein bands were visualized using ultra high sensitivity ECL kit (HY-K1005; MCE, New Jersey, USA), and images were obtained with a chemiluminescence gel imaging system (12003153; Bio-Rad, Hercules, USA).