Recombinant human tnf α

Human submandibular gland (HSG) cells were cultured as previously described (30 (link), 60 (link)) and incubated with or without 1 ng/mL human recombinant TNF-α (BioLegend, CA, USA) in serum-free medium for 24 h and subsequently lysed to isolate RNA.

The HaCaT cell line was obtained from the laboratory of Wonkwang university (Prof. Min Cheol Park). The HaCaT cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and streptomycin (Welgene, Seoul, Korea). The HaCaT cells were cultured at 37°C in an incubator with a humidified atmosphere of 5% CO₂ and 95% air. DMEM and FBS were purchased from GIBCO BRL (Grand Island, NY, USA). penicillin and streptomycin were purchased from Welgene (Seoul, Korea). Recombinant human TNF-α, IFN-γ, and IgE mouse ELISA kit were obtained from BioLegend (San Diego, CA, USA). Primary antibodies for Nrf2, Lamin B, HO-1, β-actin, and secondary antibodies used in the western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dexamethasone (#D2915), and DNCB were purchased from Sigma-Aldrich. (St. Louis, Mo., USA).

Recombinant human TGF-β, recombinant FGF-2 and recombinant human TNF-α were purchased from Biolegend (San Diego, CA). Rapamycin was from ENZO life science (Loerrach, Germany). Bafilolycin A1 was from Invivogen. FITC-PHA was from Vector (FL-1111). FITC-PNA was from Sigma (L7381). DAPI was from Sigma (D9542). Alexa Flour 488, 594 or 647 conjugated anti-mouse or anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The primary antibodies against JLP (ab12331, 1:1000), Fsp-1 (ab197896, 1:200), α-SMA (ab124964, 1:1000), Fibronectin (ab45688, 1:500), Collagen-I (ab34710, 1:1000), Ki67 (ab16667, 1:250), TGF-β (ab92486, 1:500), phospho-Smad2 (ab188334, 1:1000), phospho-Smad3 (ab52903, 1:1000) and p62 (ab56416, 1:200) were all from Abcam (Cambridge, MA). Anti-LC3 antibody (ab51520, 1:100, Abcam) was used for immuno-staining and Anti-LC3 antibody (L7543, 1:1000, Sigma) was used for western blotting, respectively. Anti-Nephrin and anti-F4/80 (123120, 1:100) antibodies were from Progen and Biolegend, respectively. Anti-Caspase-3 (#9664, 1:1000), anti-Beclin 1 (#3738,1:1000) was from Cell signaling Technology (Danvers, MA). Anti-GAPDH (sc-365062, 1:2500) was purchased from Santa Cruz (Santa Cruz, CA).

The binding activity of antibody to its antigen, recombinant human TNFα (BioLegend, San Diego, CA, USA), was measured by using 96-well Nunc MaxiSorp™ Flat-Bottom plate (Thermo Fisher Scientific). First, 100 μl TNFα solution was coated with a concentration of 100 ng/mL in sodium carbonate buffer (pH 9.6) overnight at 4  °C. The wells were washed three times with PBS-Tween solution (PBS buffer with 0.05% Tween 20) and blocked with blocking buffer (PBS-Tween containing 5% skim milk) at room temperature for 1 h. After washing three times with the PBS-Tween solution, 100 μl of adalimumab sample in serial dilution was added to each well and incubated at room temperature for 1 h. Then, the wells were washed again three times with PBS-Tween solution and incubated with 1:8000-diluted Anti-IgG (H + L chain) (Human) pAb-HRP antibody at room temperature for 1 h. The plate was washed four times with PBS-Tween solution and incubated with 100 μl ELISA POD Substrate TMB Solution (Nacalai) at room temperature. The reaction was stopped by adding an equal volume of 1 M H₂SO₄, and the absorbance was measured at 450 nm using Multiskan FC microplate reader (Thermo Fisher Scientific).

Glioma cells were treated with recombinant human TNFα (100 ng/ml) (Biolegend; Cat no. 570108) and harvested after 24 h for mRNA expression and promoter-activity analysis in pGL3F1-transfected cells.

PTX (cat: P1784) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Human TGF-β1 (cat: 580704) and recombinant Human TNF-α (570104) were obtained from Biolegend (San Diego, CA, USA). All stock solutions were diluted in culture media prior to use.