were plated at 160,000 cells ml in 75 cm2 flasks for 24 hours, then treated with vehicle or 10 uM FAK inhibitor PF573228 in vehicle for 4 hours. C6 cells were fi ed with 1% formal dehyde for 10 minutes at room temperature and then washed with and resuspended in ice cold PBS sup plemented with a protease inhibitor cocktail. Cells were scraped and centrifuged at 4 C for kinase inhibitor MG132 5 minutes at 2,000 rpm, after which the cell pellet was resuspended in 1 SDS lysis buffer and left on ice for 10 minutes. Chromatin was sheared by sonication on ice to an average size of sheared chromatin of 500 bp and up to 1. 5 2 Kbp. Sonicated samples were centrifuged for 10 minutes at 14,000 rpm at 4 C to remove any debris, and the supernatant was divided into 200 ul al iquots containing material from 1 million cells for each ChIP analysis, and then snap frozen and stored at ?80 C.

ChIP grade rabbit polyconal antibodies were against STAT3 or for normalization, Histone H3. Normal rabbit IgG was used as a control for non specific binding. Immunoprecipitation was performed according to manufacturers protocol. Chromatin precipi tated DNA was resuspended in a final volume of 40 ul of water and 1 10th of each was used for the PCR amplification. Reactions were prepared in a final volume of 20 ul with 1 PCR buf fer, 200 uM dNTP, 1. 5 mM MgCl2, 0. 5 uM each forward and reverse primers, 1 10 chromatin immunoprecipitated DNA sample and 0. 5 U of Taq DNA polymer ase. The PCR cycle used were 3 minutes at 94 C for the initial denaturation, 36 cycles of 45 seconds of 94 C, 30 seconds at 60 C, 60 seconds at 72 C, followed by 10 minutes at 72 C.

ChIP amplification products were se quenced at the University of Louisville DNA Core Facility. Western blotting Protein lysate from cell cultures was isolated using RIPA buffer supplemented with 1 mM sodium orthovanadate, 5 mM sodium fluoride and 0. 1% protease inhibitor cocktail. Cells were washed in ice cold PBS before cells were scraped from the surface with an inverted p1000 pip ette tip in RIPA buffer. Lysate was transferred to Eppendorf tubes and placed on ice. The lysate was then triturated using a 1 ml syringe and 26? gauge needle before samples were returned to ice and incubated for 30 minutes. Samples were centrifuged at 12,300 rpm at 4 C for 15 minutes.

Lysate was then transferred to fresh Eppendorf tubes and stored at ?80 C or prepared for pro tein quantitation with Pierces BCA protein assay as per manufacturers instructions. Proteins were separated by SDS PAGE and blotting was then performed with speci fic antibodies for CNTF, or with FAK, pFAK Tyr397, JNK, pJNK Tyr185, pSTAT3 Ser727, Cilengitide pSTAT3 Tyr705. Concentration used was 1 1000 STAT3, Tubulin, till all from Cell Signaling Technology. Briefly, after transfer, PVDF membranes were blocked in 5% non fat milk in Tris buffered saline with 0. 05% tween for 1 hour then incubated overnight in primary anti body. Blots were washed with TBST before incubation with appropriate Horse Radish Pero idase conjug