*4.10. Western Blot Analysis*

Total proteins were extracted with a RIPA buffer (Sigma Chemical Co., St. Louis, MO, USA). The mitochondria and cytosol fractions were isolated using a Mitochondria Isolation Kit (Abcam, Cambridge, MA, USA) following the manufacturer's procedure. Western blotting was conducted as describe in our previous report [24]. Briefly, 25 μg of total extracted proteins were separated via 10–12% SDS-PAGE prior to nitrocellulose membranes transfer by electroblotting, which was then blocked with 5% skimmed milk for 1 h. Blots were then incubated with specific antibodies HO-1 (1:200), Bax (1:200), Bcl-2 (1:200), cytochrom C (1:200), β-actin (1:500), and Cox IV (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and caspase-3 (1:500) (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 ◦C;. The horseradish peroxidase-conjugated antibodies were regarded as the secondary antibody. The bands were detected by chemiluminescence using the ECL Western blotting assay kit (Life Technologies, Seoul, Korea) and visualized by Davinch-Chemi Imager™ (CAS-400SM, Core Bio, Seoul, Korea).

#### *4.11. Data Analysis*

SigmaPlot® 12.0 (Systat Software Inc., San Jose, CA, USA) software was used to perform statistical analysis of the data. All experiments are expressed as mean ± standard deviation (SD). Student's *t*-test was performed, and statistical significance was assigned in accordance with *p* < 0.05.

#### **5. Conclusions**

The findings of this study demonstrated the cytoprotective activity of the BAPs identified as FTVN and EPTF from blue mussel protein and their role in preventing oxidative stress-mediated endothelial dysfunction (ED). Investigations of the cytoprotective mechanism of these two peptides and their combination in H2O2-mediated HUVEC injury revealed that BAPs alleviated HUVEC injury through enhancement of the antioxidant defense system and anti-necrotic action. As a result, BAPs derived from blue mussel protein might be an alternative approach in preventing CVD through protecting cardiovascular vein endothelial cells. Furthermore, to develop a nutraceutical or pharmaceutical component based on this result, more in vivo research is required.

**Author Contributions:** I.T.S. and C.-B.A. methodology, writing, conceptualization, data investigation and analysis. J.-Y.J. editing, supervising, designing, and directing the project: funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A3047702).

**Data Availability Statement:** The original contribution presented in this study are included in the article, further inquiries can be directed to the corresponding author.

**Conflicts of Interest:** The authors declare that there are no conflict of interest.

#### **References**

