Activation states of STAT3 and STAT1 in Sko-007 and U266 cells in the absence or presence of IL-6 or IFNα. 2 × 106 Sko-007 or U266 cells were left untreated or treated with IL-6 (10 ng/ml) or IFNα (100 U/ml) for 10 min and lyzed in cell lysis buffer. Then 30 μg cytoplasmic proteins were separated by 10% polyacrylamide-SDS gel and transferred elechtrophoretically to Immobilon. After blocking with 5% dry milk, the membranes were blotted with anti-STAT3, STAT1, phospho-STAT3 or phospho-STAT1 antibodies and the relevant HRP-labeled second antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham, USA).