Ta 09

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).

Total RNA of cells/tissue was isolated by TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's instructions. cDNA was then synthesized from total RNA via reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA). Next, qRT–PCR was performed using a PerfectStart Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) on a SYBR-Green real-time PCR platform (Thermo Fisher Scientific, CA, USA). Specific primers for the Kv channels are presented in Tables 1 and 2. β-Actin and U6 was used as the internal controls. The individual result was the average of 3 repeated assays for each experiment. The relative expression of each group was analyzed using the 2^-ΔΔCt method. Proteins were extracted from ventricular myocytes of mouse models and H9C2 cell lines and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were probed with primary antibodies against Kv2.1 (19963-1-AP, NM_004975, Proteintech) and β-actin (TA-09, Zhong Shan Jin Qiao Biotechnology, Shanghai, China) followed by incubation with goat anti-rabbit (7074S, Cell Signaling Technology) and mouse (SA00001-1, Proteintech) IgG, horseradish peroxidase- (HRP-) linked secondary antibody, as required. β-Actin was used as an internal control.