Quantification of Activated STAT3 Pathway

Activation of the STAT3 pathway was assessed by quantifying pSTAT3^tyr705 using immunoblot analysis with detection of fluorescent signals using an infrared fluorescence scanner (Licor Biosciences, Lincoln, NE) or with detection using an ECL chemiluminescent substrate (Amersham Biosciences, Piscataway, NJ) captured onto x-ray film (Fuji Medical systems, Stamford, CT) as described previously [12] (link), [19] (link). Briefly, following incubation with primary antibodies (anti-phospho-STAT3^tyr705[1∶500]), blots were washed with phosphate buffered saline with 0.1% Tween 20 and incubated with fluorescent-labeled secondary antibodies (1∶2500) for 1 h prior to scanning by Licor or using a Personal Densitometer (Molecular Dynamics, Sunnyvale, CA).