Horseradish peroxidase linked anti rabbit igg

They were performed as described in [25 (link)]. Briefly, cells were washed twice with ice-cold 1X PBS and proteins were extracted with 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40 supplemented with 50 mg/ml leupeptin, 50 mg/ml aprotinin, 0.5 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl fluoride (Sigma). Breast tumor samples were homogenized in the same lysis buffer with a Polytron homogeneizer. Protein extracts were quantified using bicinchoninic acid protein assay reagent (Pierce), resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies recognized human specific forms of GAPDH (Abcam) and phospho-STAT3 (Y705) (R&D). Secondary antibodies included horseradish peroxidase-linked anti-rabbit IgG and anti-mouse IgG (Amersham GE Healthcare). Proteins were detected with Immobilon western chemiluminescent HRP substrate (Millipore). Signals in Western blots were quantified with the software ImageJ 1.38 (NIH, Bethesda, MD, USA).