Western Blot Analysis of Signaling Pathways

The procedure of Western blot analysis was descried as previously [10 (link), 13 (link)]. In brief, the equal amounts (50 μg/lane) were extracted from the calf muscle tissue or HUVEC and separated by SDS-PAGE using 10~15% acrylamide gradients. The membranes were incubated with antibodies against Phosphoinositide 3-kinases (PI3K), Phospho-PI3K (1:1000; Cell Signaling, Danvers, MA, USA), AKT, Phospho-AKT (1:1000, Invitrogen, Thermo Fisher Scientific, MA, USA), STAT3 (1:1000; Abcam, Cambridge, MA, USA), Phospho-STAT3(Tyr705) (1:1000; Cell Signaling, Danvers, MA, USA), Matrix metallopeptidase 9 (MMP9) (1:1000, Arigo Biolaboratories, Taiwan), vascular endothelial growth factor (VEGF) (1:1000, Proteintech, Rosemont, IL, USA), VEGFR2 (1:1000) and Phospho-VEGFR2 (1:5000 Abcam, Cambridge, MA, USA). Signals were detected with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA). Immunoreactive bands were detected by enhanced chemiluminescence (ECL; Thermo Fisher Scientific, CA, USA) and then were exposed to ECL-Western blotting system (AVEGENE CHEMX 400). The intensity of the protein band was quantified by Image J software (Bethesda, NIH, MD, USA) and the results are expressed as normalized ratio to housekeeping gene GAPDH.