Proteins of knee cartilage from each group were extracted and collected using RIPA Lysis Buffer (KGP250, China), mixed with 10 μl phosphatase inhibitor, 1 μl protease inhibitor, and 5 μl 100 mM PMSF. Protein concentrations of each group were determined by the BCA kit (KGA902, China). Then, electrophoresis was performed, blocking with 10% milk powder for 2 h, incubated with primary antibodies anti-iNOS (diluted 1:500 affinity AF0199), anti-COX2 (diluted 1:1000 UK Abcam plcab 179800), VEGF-A (diluted1:500, UK Abcam plc ab1316), VEGFR2(diluted1:1000, UK Abcam plc ab39638). After washing again, secondary antibodies were added and incubated for 2 h at room temperature. Image-J software was used to analyze the gray scale.

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