Anti nf κb

HDF cells (1 × 10⁴ cells/well) were seeded in a chamber slide and incubated in the complete medium overnight. Then, the cells were treated with ZLE or tricin at the indicated concentrations in the complete medium for 24 h and then irradiated with 20 J/cm² UVA. The cells were fixed with 4% paraformaldehyde (w/v) at 4 °C overnight and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 2% goat serum in PBS, the cells were incubated with anti-NF-κB (1:500, Thermo Fisher Scientific) in 2% goat serum in PBS at 4 °C overnight. After washing, the cells were incubated with Alexa Fluor 488 goat antirabbit IgG (Invitrogen) for 1 h at room temperature. The slide was mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The images were obtained using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany).

The small intestine samples extracted from the mice were homogenized by centrifugation at 12,000 g for 20 min at 4°C, and the total protein from the homogenized tissue was extracted using a Solarbio Whole Protein Extraction Kit. The total concentration of protein was determined using a BCA Protein Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). For Western-Blot analysis, 50 μg of the protein extracts were loaded onto a 10% NuPAGE gel and run to isolate the proteins, and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk by shaking the membranes at 28°C and 50 rpm for 1 h. Then, the membranes were incubated with primary antibodies (anti-iNOS, anti-eNOS, anti-NF-κB) (Thermo Fisher Scientific Co., Ltd., Shanghai, China) overnight at 4°C. The PVDF membranes were washed 5 times with 1 × TBST for 5 min each, after which they were incubated with HRP-labeled diantibodies (Thermo Fisher Scientific Co., Ltd., Shanghai, China) for 1 h and washed five times with 1 × TBST for 5 min each. Finally, the PVDF membranes were stained with an ECL chemiluminescence solution to observe antibody binding, which was quantified using the ImageJ (National Institutes of Health) software package.