Rophotometrically as shown in our study [29,30]. Glutathione peroxidase (GPX) activity was detected by a industrial kit (A005-1-2; Nanjing Jiancheng Bioengineering Institute). The substrates employed for the GPX assay are hydrogen peroxide and glutathione. GPX promotes the reaction of hydrogen peroxide and decreased glutathione (GSH) to create water and oxidized glutathione (GSSG). By measuring theAntioxidants 2021, 10,4 ofconsumption of reduced glutathione within the enzymatic reaction, the activity of glutathione peroxidase is often calculated. qPCR assays have been performed after our published protocol [29,30]. The gene-specific primers are presented in Supplementary Supplies Table S3. The 2-Ct technique was made use of for the quantification of qPCR by utilizing TATA-box-binding protein (tbp) and glyceraldehyde3-phosphate dehydrogenase (gapdh) as two reference genes. The relative abundance of gene expression was normalized towards the A-Se group (as “1”). 2.three.four. Western Blot Evaluation In the present study, we detected the protein expression of SELENOS, SELENOM, SELENON, GRP78, SREBP-1c, and ACC on account of their value in ER anxiety function and lipogenic metabolism, and also resulting from availability of well-tested reagents (e.g., antibodies), and also the protocols were described earlier [29,30]. Total protein was isolated working with RIPA buffer (P0013B; Beyotime Biotechnology, Shanghai, China), and nuclear and cytoplasmic protein extraction had been performed using a commercial kit (P0027, Beyotime Biotechnology). Protein extracts had been separated via 85 sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes had been subsequently 5-HT7 Receptor Antagonist manufacturer blocked in five defatted milk and incubated with principal antibodies overnight at four C. We made use of antibodies against SELENOM (ab133681; Abcam, London, UK), SELENON (55333-1-AP; Proteintech Group, Wuhan, China), SELENOS (15591-1-AP; Proteintech Group, Wuhan, China), GRP78 (sc-166490; Santa Cruz, CA, USA), SREBP-1c (ER1917-19; Huabio, Hangzhou, China), ACC (21923-1-AP; Proteintech Group, Wuhan, China), GAPDH (10494-1-AP; Proteintech Group, Wuhan, China), and IgG Adenosine A3 receptor (A3R) Antagonist medchemexpress antibody (#7074; Cell Signaling Technology, Boston, MA, USA) to detect the expression with the corresponding proteins. The protein bands were visualized by the instrument of Vilber FUSION FX6 Spectra imaging system (Vilber Lourmat) and then quantified by the software Image-Pro Plus six.0 (Media Cybernetics). two.3.5. Immunofluorescence Analysis We utilized the immunofluorescence to measure the distribution and expression of GRP78 protein inside the intestine depending on Zhao et al. [30]. Tissues were fixed with 4 paraformaldehyde, permeabilized with PBS containing 0.1 Triton X-100 (PBS-T), and blocked with standard goat serum. Slides were blocked for 30 min with regular goat serum and incubated overnight at 4 C with the anti-GRP78 antibody (1:200, sc-166490; Santa Cruz, CA, USA). Just after a wash step, slides were incubated with IgG H L CoraLite488 (1:1000, SA00013-2; Proteintech Group, Wuhan, China) for 1 h, and then nuclei were stained with DAPI (1 /mL, ab228549; abcam). The images had been acquired working with a laser scanning confocal microscope (TCS, SP8, Leica Microsystems, Wetzlar, Germany). two.3.six. Assays for Luciferase Activities Assays for the luciferase activities had been performed just after Wei et al. [29] and Zhao et al. [30]. The relative luciferase activities were expressed as the ratio of Firefly luciferase to Renilla luciferase. two.3.7. EMSA The preparation of t.
