Anti p iκb

Total proteins were extracted using RIPA lysis buffer within 1% protease inhibitor cocktail (Millipore, Bedford, MA, USA) 0.1 mM according to the manufacturer’s instructions. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking, the membranes were then incubated at 4 °C overnight with the following primary antibodies: anti-HIF-1α, anti-VEGF-A, anti-JNK, anti-PPAR-γ, anti-C/EBP-α, anti-FABP4 (1:1000, all Cell Signaling Technology, Danvers, MA, USA), anti-NF-κB p65, anti-p-IκB, anti-TNF-α, anti-MCP-1 (1:1000, all Abcam, Cambridge, MA, USA), anti-SFRP5, anti-Wnt5a, anti-Wnt10b, anti-β-catenin (1:1000), and anti-β-actin (1:5000, all GeneTex, Irvine, CA, USA). After washing, the membranes were probed with corresponding second antibodies (1:3000, GeneTex). The density of the individual protein bands was quantified by densitometric scanning of the blots using ImageJ software.

Bovine serum albumin (BSA) was obtained from Shanghai Hengyuan Technology Biology Co., Ltd (Shanghai, China). Glucose, Phenylmethylsulfonyl Fluoride (PMSF), and ethylene diamine tetraacetic acid disodium salt (EDTA) were obtained from Beijing solabo Technology Co., Ltd. (Beijing, China). Penicillin and streptomycin were bought from Sangon Biotech Inc. (Shanghai, China). Ethanol (purity > 99%) was obtained from Xilong Chemical Co., Ltd. (Guangdong, China). Fetal bovine serum (FBS) was bought from Sangon Biotech, Inc. (Shanghai, China) and Dulbecco’s modified eagle medium (DMEM) was purchased from ThermoFisher (MA, USA). CCK-8 kit was obtained from Shanghai Yanjin Biotechnology Co., Ltd. (Shanghai, China). Anti-p-P38, anti-P38, anti-JNK, anti-p-JNK, anti-NF-κB p65, anti-p-NF-κB p65, anti-IκB, anti-p-IκB, anti-AKT and anti-p-AKT antibodies were purchased from Abcam Technology (Cambridge, UK). Anti β-actin antibody, Goat anti-rabbit and mouse IgG and mouse anti-goat secondary antibodies were purchased from ZSGB Biotech Co., Ltd. (Beijing, China). Unless specified, all other reagents are obtained from Sigma Chemical Co. (St. Louis, MO, USA).

All cells were collected and lysed in RIPA buffer (Beyotime, China), and the whole proteins, nucleoproteins, and cytoplasmic proteins were extracted as required. Protein concentrations were determined by BCA protein assay kit (KeyGEN BioTECH, China). Equal quantities of protein were separated on 12% SDS–PAGE, electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, USA), and then blocked with 5% non-fat milk in TBST buffer for 2 h at room temperature. The membranes were then incubated with the corresponding antibodies overnight at 4 °C. The corresponding antibodies were: anti-IκB, anti-pIκB, anti-TLR4, and anti-NF-κB(Abcam, Cambridge, UK). Samples were then washed three times and incubated with the horseradish peroxidase-conjugated secondary anti-rabbit/mouse antibody for 1 h at room temperature. The proteins were visualized using an enhanced ECL detection kit (Dingguo changsheng biotechnology CO., Ltd., China) and scanned with a Clinx ChemiScope chemiluminescence imaging system (ChemiScope 5300 Pro). The relative optical densities of specific proteins were estimated utilizing a ChemiScope analysis program.