3.3.5. Western Blot Analysis

To investigate the e ffect of LJSF4 on the expression of apoptosis-related proteins, Western blot analysis was performed by the method described by Wijesinghe et al. [47]. Briefly, cells exposing to 30 mJ/cm<sup>2</sup> of UVB were pretreated with di fferent concentrations of LJSF4. After incubation for 24 h, cells were harvested in lysis bu ffer. The total protein levels of the supernatant were measured using a BCATM kit. A gradient 10% SDS-PAGE was used to separate the total protein extracts (50 μg), which were then electrophoretically transferred onto nitrocellulose membranes. The membranes were then blocked with skim milk (5%), and the immunoblotting procedure, including incubation with primary antibodies, washing, and incubation with secondary antibodies, was carried out as previously reported [47]. Finally, the target bands were observed by enhanced chemiluminescence (ELC), and detected by Tanon-5200 detection system (Tanon Science, Shanghai, China).

## *3.4. E*ff*ect of LJSF4 on UVB-Irradiated Zebrafish*

## 3.4.1. Origin and Maintenance of Parental Zebrafish

This study work was approved by the ethical committee of experimental animal care of Ocean University of China at College of Food Science and Engineering, (Approval No. 2019-05). Adult zebrafish were obtained from the Molecular Medicine Laboratory (Ocean University of China, Qingdao, China) and kept in an automatic circulation culture system (ESEN, Beijing, China) at 28.5 ± 1 ◦C with a cycle of 14/10 h of light/dark. Live brine shrimps were fed twice a day to the parental zebrafish. Embryos were obtained from natural spawning according to the method reported by Zou et al. [48]. Fertilized embryos were collected in petri dishes completely within 30 min.

#### 3.4.2. Measurement of E ffect of LJSF4 Against UVB-Irradiation in Zebrafish

At 2 dpf, the zebrafish were transferred to a 24-well plate and treated with LJSF4 with final concentrations of 25, 50, and 100 μg/mL, respectively. The incubation, UVB-irradiation process and determination of ROS level, cell death, and NO production were carried out according to the reported procedure of Ko et al. [12]. The zebrafish larvae were photographed under the microscope Cool SNAP-Procolor digital camera, and the individual zebrafish larvae fluorescence intensity was quantified using an Image J program (National Institutes of Health, Bethesda, MD, US).
