Recombinant human tnf α

Chitin beads, the restriction enzymes and the plasmid pKYB-MCS were purchased from New England Biolabs (NEB, Ipswich, USA). E. coli strain ER2566 was kept in our laboratory. T4 DNA ligase was obtained from TaKaRa (Dalian, China). Synthetic peptides were purchased from Sinoasis Pharmaceuticals (Guangzhou, China). Primer synthesis and DNA sequencing were performed by Invitrogen Company (Guangzhou, China). Human mammary non-tumorigenic epithelial cells MCF10A, HUVEC cells, cervical cancer HeLa cells, leukemia K562 cells, breast cancer MCF-7 and MDA-MB-231 cells, esophageal cancer Eca109 cells, prostate cancer Du145 cells, lymphoma Raji cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Recombinant human TNF α, TNFRI and TNFRII were purchased from Sino Biological Inc. (Beijing, China). Human and mouse serum albumin (HSA and MSA) were purchased from Sigma (Louis, USA). P21 Rabbit mAb, CyclinA Rabbit mAb, CyclinD Rabbit mAb, CyclinE Rabbit mAb and were purchased from Cell Signaling Technology (Beverly, USA). P27 Mouse mAb and E2F1 Mouse mAb was respectively purchased from Abcam (Cambridge, USA) and NeoMarkers (Fremont, USA).

Recombinant human TNF-α (Sino Biological Inc., China); curcumin (Sigma-Aldrich, St. Louis, MO, USA); pre-stained protein standards (Fermentas, Lithuania); Human Annexin V Apoptosis Detection Kit and cell cycle test kits (BD Biosciences, San Jose, CA); Bradford Assay kit (BD Biosciences, San Jose, CA). Antibodies against NF-κB-p65, phospho-NF-κB-p65, caspase-8, caspase-9, ERK, phospho-ERK (pERK), Akt andphospho-Akt (pAkt) (Cell Signaling Technology, Boston, MA, USA); β-actin antibody (Santa Cruz Biotechnology, USA); Nuclear and Cytoplasmic Protein Extraction Kit, LDH Cytotoxicity Assay Kit and CCK-8 Assay Kit (Beyotime, CHN); ECL detection kit (Cell Signaling Technology, Boston, MA, USA). Water was ultra-pured by a Milli-Q water purification system (Millipore, USA).

CC cell lines (SW480/SW620) were purchased from the American Type Culture Collection (ATCC, USA), cultivated within L15 media that contained 10% FBS (VivaCell, Shanghai, China) and incubated in the ventilated environment under 37 °C and 95% air and 5% CO₂ conditions. Recombinant human TNF-α was obtained from Sino Biological, and recombinant human CXCL10 was purchased from Peprotech. At the final concentrations listed, the following basic pharmacological inhibitors were used: SB203580 (the inhibitor of p38 MAPK, Selleck), LY294002 (the inhibitor of PI3K, Selleck), MK-2206 2HCI (the inhibitor of Akt, Selleck), BAY11-7082 (the inhibitor of NF-κB, Selleck) and CHIR-99021 (the inhibitor of GSK-3β, MCE). All the cellular studies were carried out under specified and standard conditions.