Hrp labeled anti rabbit igg

Cytosolic and mitochondrial fractions were separated on 14% SDS-polyacrylamide gels under reducing conditions and electrotransferred to PVDF membranes (Immobilon, Millipore, Billerica, MA; 0.45 μm pore; 400 mA, 1.5h) using CAPS buffer, as above. Membranes were blocked with 5% nonfat milk in TBST, and subsequently immunoreacted with mouse monoclonal anti-CytC antibody (BD; 1 μg/ml in 5 % nonfat milk in TBST, overnight, 4°C) followed by HRP-labeled anti-mouse IgG (1:10,000; GE Healthcare), and ECL detection, as above. As loading controls, membranes were probed with rabbit polyclonal anti-β actin (Novus Biologicals; 1 μg/ml, overnight, 4°C) followed by HRP-labeled anti-rabbit IgG (1: 5,000; GE Healthcare). Densitometric assessment of band intensities was performed using ImageJ software (rsbweb.nih.gov)

D5-HER2 cells were lysed using radio-immunoprecipitation assay (RIPA) buffer (Boston Bioproducts; Ashland, MA) and protein concentration was determined by the BCA assay (Bio-Rad). Approximately 16–20μg of protein was run on tris-glycine gels under reducing conditions. Protein was transferred to a nitrocellulose membrane and blocked for two hours using non-fat milk (Bio-Rad). The following primary antibody dilutions were used: Stat5a (ab32403; 1:1000; Abcam; Cambridge, England, United Kingdom), anti-phospho Y694 Stat5a (ab30648; 1:500; Abcam), GAPDH (D16H11; 1:1000; Cell Signaling; Danvers, MA). Primary antibodies were diluted in PBS-T +5% non-fat milk and incubated overnight at 4°C. Membranes were incubated for one hour at room temperature with secondary antibody (HRP labeled anti-rabbit IgG; 1:10,000; GE Healthcare; Little Chalfont, Bucking hamshire, United Kingdom) diluted in PBS-T. For Stat5a and phospho-Stat5a westerns, Supersignal West Femto high sensitivity substrate (Thermo Scientific) was used for visualization while Supersignal West Pico (Thermo Scientific) was used for GAPDH westerns.