Irdye 800 labeled goat anti mouse igg

Membranes were probed using mouse-derived anti-phospho-STAT3 antibody from Cell Signaling Technologies, USA, and anti-β-actin from Sigma, USA. Bands were detected with IRDye 800-labeled goat anti-mouse IgG (LI-COR Biosciences, USA) and imaged using an Odyssey infrared imaging system (LI-COR Biosciences, USA).

Transfected cell cultures were lysed in buffer containing 0.1M Na-phosphate (pH 7.4, 0.2% Triton X-100, 0.1 mM EDTA, 0.2 mM PMSF, 1 μg /ml aprotinin and 1 μg /ml leupeptin) by three-repeated freezing and thawing cycles. The resulting cell lysates were centrifuged at 13,000 g for 10 min. Proteins were separated by SDS-PAGE (12% acrylamide) and electro-blotted on nitrocellulose paper for 1 hrs at 180mA. The nitrocellulose was blocked overnight with 10% no-fat milk in Tris buffered saline (TBS). The primary antibodies were diluted in 5% no-fat milk in TBS and incubated with the nitrocellulose for 1.5 hrs. The membranes were rinsed in TBS-tween 20, and incubated with the secondary antibodies (IRDye 800-labeled goat-anti- mouse IgG, LI-COR Biosciences, Lincoln, NE; diluted 1:15,000) in 5% non fat milk in TBS for 45 min. The Odyssey infrared imaging system (LI-COR Biosciences) was used to measure protein concentration. Scanning parameters (non-saturated signals, resolution of 84 or 169 μm for high/medium quality) were set according to the manufacture’s instructions. Outlines were drawn around the bands and the integrated intensity was calculated after subtracting background. The amount of SMN protein was normalized versus actin signals and compared among groups.