erformed in duplicate. Following the termination of the reaction, the slides were subjected to three10 min washes in 1X TBS/T, three-10 min washes in 0.5% SDS, and one quick rinse with distilled 17429684 water before being 
spun dry and exposed to X-ray film. Control slides were incubated with MedChemExpress KU-55933 kinase reaction mixture without kinase and processed in parallel. Exposures were taken for each microarray assayed at 10, 17, and 24 hrs. Materials and Methods Protein purification and human protein microarrays Human proteins were purified as 66-GST-His-fusion proteins from yeast using a high-throughput protein purification protocol described previously. Tissue culture preparation U373MG HGF2/C-Met+ cells were maintained in Dulbecco’s Modified Essential Medium low glucose with L-glutamine and sodium pyruvate supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as previously described. All cells were grown at 37uC in a humidified incubator with 5% CO2. U373 cells were incubated in the aforementioned media supplemented with low serum overnight and treated with 50 ng/mL HGF or PBS control for 30 min. At the end of each experiment, cells were washed and harvested in 1X PBS in preparation for protein extraction for phosphorylation assays and immunoblot analyses. Phosphorylation using U87MG, and U373MG cell lysates Protein microarrays were blocked in 1X TBS/T with 0.1% BSA for 1 hr at RT with gentle shaking. 0.2 mg/mL of total lysate was diluted into kinase buffer. Lysate-buffer mixture was then incubated on arrays and cover slipped before being placed in a humidity chamber at 30uC for 30 min. Assays were performed in triplicate. The slides were subjected to three-10 min washes in 1X TBS/T, three-10 min washes in 0.5% SDS, and one quick rinse with distilled water before being spun dry and exposed to X-ray film. Control slides were incubated with kinase reaction mixture without kinase and processed in parallel. Arrays were developed by autoradiography for a short and long exposure time. Tumor xenografts Glioma xenografts were generated as previously described from U87MG HGF+/C-Met+ cell lines originally obtained from the American Type Culture Collection. Cells were grown in MEM with Earle Salts and L-glutamine supplemented with 10% fetal bovine serum, 2 mmol/L sodium pyruvate, 0.1 mmol/L MEM-nonessential amino acids, and penicillinstreptomycin at 37uC in a humidified incubator with 5% CO2. Female 6- to 8-week-old mice were anesthetized by i.p. injection of ketamine and xylazine. For subcutaneous xenografts, Nu/nu mice received 46106 cells in 0.05 mL of plain media s.c. in the dorsal flank. When tumors reached,200 mm3, the mice were randomly divided into groups and received the indicated doses of either L2G7 or isotype matched control mAb in 0.1 ml PBS i.p., as previously described. Tumor volumes were estimated by 22634634 measuring two dimensions and calculating volume as V = ab2/2 Protein microarray data acquisition After lysate assays were conducted on the protein microarrays and exposed to X-ray film, the film was scanned at 4,800 dots per inch. Digitized images in TIFF format were then analyzed using GenePix Pro 6.0 and labeled Phospho-X. Post phosphorylation, each array was subsequently probed with antiGST antibody, digitized with a GenePix 4000 scanner, analyzed using GenePix Pro 6.0, and labeled Anti-GST-X. Positive and negative control proteins Profiling the Human Phosphorylome were imprinted on each array as markers to properly align the grids a