Anti wnt5a

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.