Anti py stat3

Cells were lysed in lysis buffer (0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40 and 1× PBS) containing proteinase inhibitors (100 µg/mL phenylmethylsulfonyl fluoride (PMSF), 13.8 µg/mL aprotinin (Sigma), and 1 mM sodium orthovanadate (Sigma). Total cell lysates were incubated on ice for 30 min, followed by microcentrifugation at 10,000 g for 10 min at 4°C. Protein concentrations of the supernatants were determined by the Bio-Rad method. Equal amounts of protein (10 µg) were mixed with 5× SDS sample buffer, boiled for 5 min, and separated by 8 or 12% SDS-PAGE, and transferred onto PVDF membranes (0.45 µm; Millipore). Intracellular and secreted hormones were determined using polyclonal antisera to PRL or GH (NHPP) applied at dilutions of 1:8000 and 1:50,000, respectively. Immunoblotting was performed using anti-NG2 (Millipore, 1:1000), anti-pY-STAT3 (Y705, 1:1000), STAT3 (1:2500), and tubulin (1:1000) (all from Cell Signaling). Nonspecific binding was blocked with 5% nonfat milk in 1× Tris-buffered saline (TBST with 0.1% Tween-20). After washing for 3× 10 min in 1× TBST, blots were exposed to the secondary antibody (anti-rabbit IgG-HRP, Santa Cruz) at a dilution of 1:2000 and were visualized using ECL chemiluminescence detection system (Amersham).