Anti cxcr4 umb2

Western blots were performed as previously described (12 (link)). Briefly, cells were lysed in keratin extraction buffer (1% Triton-X 100, 0.02 mM Tris, 0.6 M KCl, and 1 mM PMSF, pH 7.0) and protein concentrations were determined by bicinchoninic acid (BCA) assays (Beyotime, Nantong, China). Proteins were separated in SDS-PAGE 12.5% gels and blotted onto PVDF membrane (Millipore). After incubation for 1 h in blocking buffer (Tris-buffered saline with 5% nonfat milk), the membrane was incubated with primary antibodies overnight at 4°C, followed by a further incubation with HRP-coupled secondary antibodies at room temperature for 2 h. Signals were visualized with an enhanced chemiluminescent kit (GE Healthcare, Chicago, IL, USA). The following antibodies were used: Mouse monoclonal anti-JWA (contract produced by AbMax, Beijing, China) and anti-GAPDH (6C5; Beyotime); rabbit polyclonal anti-Flag (Beyotime); rabbit monoclonal anti-CXCR4 (UMB2, Abcam); Rabbit monoclonal anti-AKT (C67E7) and anti-pAKT (D25E6; Cell Signaling Technology, Inc., Danvers, MA, USA); and HRP-coupled polyclonal goat anti-mouse or rabbit IgG (Beyotime).