Tnf α recombinant protein

We purchased the following antibodies including RelA/ p65 (NF-κB) (#3033T; Lot# 17), phospho-IκBα (Ser32/36) (#9246S; Lot# 16), p-JNK (Thr183/Tyr185) (#9251S; Lot# 11), E-cadherin (#5296S; Lot# 2), N-cadherin (#4061S; Lot# 3), Slug (#9585S; Lot# 2), and MMP-9 (#2270S; Lot# 2) from Cell signaling Technology (Danvers, MA, USA). Antibodies against phospho-c-jun (Ser63) (sc-822; Lot # L0717), Twist1 (sc-15393; Lot # F1109), TIMP3 (sc-373839; Lot # D2316), actin (sc-1616; Lot # L3004) and lamin A (sc-7292; Lot # L1919) were obtained from Santa Cruz Biotech Inc. (Dallas, TX, USA). The c-fos antibody (GTX129846; Lot # 42256) was purchased from GeneTex Inc (Irvine, CA, USA). Sodium bicarbonate, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). We also purchased fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), NE-PER nuclear and cytoplasmic extraction reagent Kit and sodium-dodecyl sulfate (SDS) from Thermo Fisher Scientific (Waltham, MA, USA). Human tumor necrosis factor-α (TNF-α) recombinant protein was purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Mixed glial cultures were prepared from cerebral cortices of 4-day-old C57BL/6J mice as previously described [16 (link)]. Briefly, meninges and blood vessels were removed, and forebrains were dissociated by repeated pipetting in DMEM/F12 medium (Fisher Scientific, Madrid, Spain). Cells were seeded in DMEM/F12 with 20% FBS at a density of 3 × 10⁵ cells/mL and maintained at 37 °C in humidified 5% CO₂/95% air. Confluence was reached at 10–12 days in vitro and cultures were treated with SAA1 recombinant protein (R&D Systems, Minneapolis, MN, USA), TNF-α recombinant protein (R&D Systems, USA), IL-1β recombinant protein (R&D Systems, USA), LPS (Sigma-Aldrich, Madrid, Spain), and TAK242 (Sigma-Aldrich, Madrid, Spain) in DMEM/F12 with 10% FBS.