Horseradish peroxidase conjugated antibody to rabbit igg

Western blot analysis was performed as previously described 23. Cells were washed twice with cold PBS, harvested, and then sonicated in lysis buffer containing 10 mM Tris–HCl buffer (pH 7.5), 1% SDS, 1% Triton X‐100, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, Rockford, IL). Proteins in the supernatant were separated by electrophoresis on 10% SDS‐polyacrylamide gels and transferred to a PVDF membrane. We detected expressions of TRAP, NFATc1, IκBα, uPA, uPAR, GAPDH, phospho‐AMPK, AMPK, phospho‐Akt, Akt by using anti‐TRAP antibody (Santa Cruz Biotechnology, Dallas, TX), anti‐NFATc1 antibody (Santa Cruz Biotechnology), anti‐IκBα antibody (IMGENEX, San Diego, CA), anti‐uPA antibody (Santa Cruz Biotechnology), anti‐uPAR antibody (Santa Cruz Biotechnology), anti‐GAPDH antibody (Sigma–Aldrich), anti‐phospho‐AMPK antibody (Cell Signaling Technology, Danvers, MA), anti‐AMPK antibody (Cell Signaling Technology), anti‐phospho‐Akt antibody (Cell Signaling Technology), anti‐Akt antibody (Cell Signaling Technology) followed incubation with horseradish peroxidase‐conjugated antibody to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).

Western blot analysis was performed as previously described 30. We detected expressions of uPAR, TRAP, NFATc1, GAPDH, phospho‐Akt, or Akt by using anti‐uPAR antibody (Santa Cruz Biotechnology, Dallas, TX), anti‐TRAP antibody (Santa Cruz Biotechnology), anti‐NFATc1 antibody (Santa Cruz Biotechnology), anti‐GAPDH antibody (Sigma–Aldrich), anti‐phospho‐Akt antibody (Cell Signaling Technology, Danvers, MA) or anti‐Akt antibody (Cell Signaling Technology) followed incubation with horseradish peroxidase‐conjugated antibody to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).