Rabbit anti p p65

TAK-242 (CAS Number: 243984-11-4; molecular formula, C₁₅H₁₇ClFN0₄S; molecular weight, 361.82) was supplied by MedChemExpress Company (Princeton, NJ, USA). Polydatin (CAS Number: 65914-17-2; purity, 95%, molecular formula, C₂₀H₂₂O₈; molecular weight 390.38) and Oil Red O (cat.no. 00625) obtained from Sigma-Aldrich (St. Louis, MO, USA). P. sibiricum polysaccharides (cat.no.A16GS145378, purity 70%) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. The mouse ICAM-1 (cat.no. JYM0003Mo) and VCAM-1 (cat.no. JYM0251Mo) ELISA kits were purchased from Wuhan ColorfulGene Biological Technology Co., Ltd. Hematoxylin-eosin Dye kit (cat.no. KGAA224), Ecl chemiluminescence kit (cat.no. KPG1121), Rabbit Anti-GAPDH (cat.no. KGAA002), and HRP-linked anti-rabbit IgG (cat.no. KGAA35) were obtained from Jiangsu KeyGEN Biotechnology Co., Ltd. Rabbit Anti-TLR4 (cat.no. 19811-1-AP) was obtained from Proteintech (Rosemont, IL, USA). Rabbit Anti-MyD88 (cat.no. ab219413), Rabbit Anti-p65 (cat.no. ab32536), and Rabbit Anti-p-p65 (cat.no. ab76302) were supplied by Abcam (Cambridge, UK). Nitrocellulose (NC) membrane (cat.no.6648) was obtained from Pall (New York, USA).

The proteins (n = 5) of bursa were extracted with RIPA lysis buffer and determined concentration with the bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China). Then, the equal amount of protein in each group was added to the SDS–polyacrylamide gel and transferred onto PVDF membranes and blocked for 1 h using 5% skimmed milk. Subsequently, the primary antibodies, including goat anti–Bax (1:1000, Biorbyt, Cambridge, UK), rabbit anti–Bcl–2 (1:1000, Biorbyt, Cambridge, UK), rabbit anti–Caspase–3 (1:1000, CST, Boston, MA, USA), rabbit anti–Nrf2 (1:1000, Proteintech Group, Inc., Wuhan, China), rabbit anti–HO-1 (1:1000, BIOSS, Beijing, China), rabbit anti–p-iκb (1:1000, Abcam, Cambridge, UK), rabbit anti–p-p65 (1:1000, Abcam, Cambridge, UK) or rabbit anti–β–actin (1:4000; Co Win Biotech Co., Inc., Beijing, China), incubated with the membranes overnight at 4 °C. Then, the membranes were washed with TBST and incubated with horseradish peroxidase–conjugated goat anti–mouse/rabbit IgG (1:8000; Co Win Biotech Co., Inc., Beijing, China) for 2 h. The target bands obtained in the blots were scanned and measured using ImageJ 4.0.2 software (Scion Corp., Frederick, MD, USA) The data were expressed as the IOD of the target bands and compared to the corresponding β–actin values. Repeat the test three times for each sample.