Figure 4

STAT3 inhibition mediates CNTF repression by FAK. A) FAK inhibition reduces STAT3 phosphorylation on its inhibitory residue (s727) as shown in western blots of C6 cells incubated for 4 hours. Total STAT3 antibodies were used as internal controls. B) Pre-incubation with a STAT3 antagonist reduced the CNTF-inducing effects of FAK inhibitor in C6 cells. Thus, FAK represses CNTF by inhibiting STAT3. An AP1 transcription factor antagonist was without effect. Data are fold compared to control (not shown) and means +/−SEM. C) STAT3 binds the CNTF promoter region of C6 cells as shown by ChIP analysis. STAT3 antibody immunoprecipitates (IP) CNTF promoter DNA as shown by the PCR amplification products of two promoter-specific primer sets. Normal rabbit IgG was used as control for non-specific binding. Histone H3 was a positive control. D) Sequencing of the amplification products of the STAT3 immunoprecipitate gave the predicted DNA regions. Key: Consensus STAT3 binding sites; : CNTF initiation site; CNTF1 primer set (FWD, REV);; < > denote DNA regions excluded from this panel for presentation purposes. E) IL-6 treatment of C6 cells for 15 minutes robustly increased phosphorylation of STAT3 at the Tyr-705 residue (y705) with modest increases after CNTF and LIF as shown by western blot. Ser-727 phosphorylation (s727) or total STAT3 (tSTAT3) was not affected. Similar results were seen at 4 hours. The blot is representative of 4 independent experiments. F) IL-6 induced only an ~10% increase in CNTF mRNA expression in C6 cells after 4 hours and did not augment FAKi-induced CNTF expression (n = 3-4 each, p < 0.05). G) FAK inhibition reduced phosphorylation of STAT3 (y705) in C6 cells most notably under IL-6 treated conditions. Antibodies against total STAT3 were used as internal controls for western blots. Results were repeatable in independent experiments.