Tnf α sc1351

Recombinant FGF10 (catalogue: 345-FG-025) was purchased from R&D Systems (Minneapolis, MN). Antibodies against FGF10 (ab115825), MAP-2 (ab5392), GFAP (ab7260), tubulin (ab179513) and t-PI3K (ab22653) were purchased from Abcam (Cambridge, MA). Antibodies against p65 nuclear factor-κB (NF-κB, sc-372), IκB (sc-371), TNF-α (sc1351) and IL-6 (sc-1265) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against p-Akt (2965), t-Akt (9272) and p-PI3K (4288) was purchased from Cell Signaling Biotechnology (Danvers, MA). Fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Promega (Medison, WI). Caspase-3, −8 and −9 activity colorimetric kits were purchased from Abcam. 2,3,5-triphenyltetrazolium chloride (TTC), wortmannin and Akt1/2-KI were purchased from Sigma Chemical Company (St. Louis, MO)

NaF was purchased from Chengdu Kelong Chemical Co., Ltd. (Chengdu, China). Reagent kits for determination of biochemical parameters were purchased from Nanjing Jiancheng Bioengineering Institute of China (Nanjing, China). RNAiso Plus, Prim-Script^TM RT reagent Kit and SYBR^® Premix Ex Taq^TM II were obtained from Takara Biotechnology (Dalian) Co., Ltd. (Dalian, Liaoning, China). Radio-immunoprecipitation Assay (RIPA) lysis buffer (P0013C) and bicinchoninic acid (BCA) Protein Assay Kit (P0012) were purchased from Beyotime Biotechnology, China. RPMI 1640 (11875119) was supplied by Gibco, UK. The mouse NF-κB (ab32536), IκB (ab32518) and COX-2 (ab179800) were obtained from Abcam, UK. IL-6 (12912), p-NF-κB (3033), and anti-rabbit IgG (7074P2) were purchased from Cell Signaling Technology, USA. TNF-α (sc-1351) and IL-10 (sc-1783) were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). IL-1β (abs115412), IL-4 (abs116760) and bicinchoninic acid (BCA) were obtained from Absin Bioscience Inc. (Absin, Shanghai, China). Anti-goat IgG antibody (BL004A) was supplied by Biosharp Bioscience Inc. (Biosharp, Anhui, China). Other chemicals including 75% ethanol, 100% ethanol, isopropyl alcohol and chloroform used in the experiment were of analytical grade.

Tissue extracts (120 μg) were resolved by 10% reducing SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes [37 (link)]. The membranes were blocked for 2 h at room temperature in 3% bovine serum albumin solution in 20 mM Tris–HCl, pH 7.4 containing 150 mM NaCl and 0.02% Tween 20 (TBST) followed by overnight incubation at 4 °C with 1:500 polyclonal anti-MMP9 (sc-6841, Santa Cruz Biotechnology), MMP3 (sc-6839, Santa Cruz Biotechnology), TNF-α (sc-1351, Santa Cruz Biotechnology), IL-1β (sc-7884, Santa Cruz Biotechnology), IL-17 (sc-374218, Santa Cruz Biotechnology), TGF-β (sc-7892, Santa Cruz Biotechnology,) and β-actin (4967S, cell signalling technology, MA, USA) antibodies. The membranes were washed four times with TBST and then incubated with their respective alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology) (1:2000) for 1.5 h. The bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution (Sigma).