Downregulation of Src or STAT3 leads to decreased STAT3 DNA binding. The human OSA cell line SJSA and canine OSA cell line OSA8 were incubated with media, DMSO, or drug (A) SU6656, B) LLL3) for 72 hours. Cells were harvested and nuclear protein isolated. Nuclear protein was added to binding reactions with labeled species specific DNA probes for the STAT3 recognition sequences located in the promoter for survivin in the presence or absence of unlabelled competitor probe. Additionally, anti-STAT3 antibody was added to nuclear protein from cells treated with media alone to demonstrate specificity of the binding reaction. Reactions were separated on an acrylamide gel, transferred to a nylon membrane, and the DNA was crosslinked. The membranes were processed using the LightShift Chemiluminescent EMSA kit.
Fossey et al. BMC Cancer 2009 9:81 doi:10.1186/1471-2407-9-81