Primary antibodies against tnf α

DEN (molecular formula: C₁₆H₂₅NO₂, MW: 263.19, and CAS No. 2115-91-5) was purchased from Chengdu Pufei De Biotechnology Co., Ltd. (Chengdu, China). Recombinant human IL-1β was purchased from PeproTech (Cranbury, NJ, USA). CCK-8 was obtained from Dojindo (Kumamoto, Japan). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA). The primary antibody against GAPDH was obtained from Abcam (Cambridge, MA, USA). The primary antibodies against Col2a1, p21, and ACAN were obtained from Abcolonal (Wuhan, China). The primary antibodies against TNF-α, MMP13, ADAMTS5, p65, β-Actin, and p16^INK4a were purchased from Proteintech (Wuhan, China). The primary antibodies against IL-6, IκB-α, and Lamin B were purchased from Abmart (Shanghai, China).

SCSP was synthesized by Wuxi MimoTopes Biotechnology (Wuxi, China) [13 (link)]. Cyclophosphamide was supplied by Hengrui Medicine (Lianyungang, China). BUN, CRE, GSH-Px, MDA, SOD, CAT determining kits and the H&E staining kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits and PAS stain kit were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The primary antibodies against β-actin, NF-κB p50, NF-κB p65, IKKα, IKKβ, and IκBα were supplied by Beyotime Biotechnology (Shanghai, China). The monoclonal antibodies against Bcl-2, Bax, caspase 3, and caspase 9 were provided by Cell Signaling Technology Inc. (Beverly, MA, USA). The primary antibodies against TNF-α were supplied by Proteintech Group, Inc. (Wuhan, China). All other reagents were analytically pure.

Gastric tissues were cut into pieces and homogenized with ice-cold cell lysis buffer for western blotting and an IP kit (Beyotime, shanghai, China) for 30 min at 4°C. The homogenates were centrifuged at 14000 g for 10 min at 4°C. Then, the supernatants were collected and the concentrations of total protein were detected by a BCA protein assay kit (Boster, Wuhan, Hubei, China). Equivalent amounts of proteins were separated by electrophoresis on 12% and 5% sodium dodecylsulfate (SDS) polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes (Beyotime, shanghai, China). The membranes were blocked with 5% BSA at room temperature for 2 hours. The blots were incubated overnight at 4°C with primary antibodies against TNF-α (1 : 1000, Proteintech Group Inc, Chicago, USA), Bax, Bcl-2, GAPDH, and NF-κB p65 (1 : 200, Boster, Wuhan, China). After washing, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Boster, Wuhan, Hubei, China) for 2 hours at room temperature. Photodensity analysis was utilized with the chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) after visualizing by HRP substrate ECL solution (Boster, Wuhan, China).