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ELISA chemiluminescent reagent was added to each well and after 1 min, the plate was read in a luminescence plate reader using a 100 ms integration time. Cells were then washed once with DMEM lacking cysteine and methionine, and the medium was replaced with DMEM noS including dialyzed serum and kinase inhibitors as indicated. After incubation for 1 h, 50 lCi of Expre35S35S was added to each well and the cells were labeled for 4 h. Cells were washed once with ice cold PBS, and lysed as described above for Western blotting.

Following separation by SDS PAGE, and transfer to nitrocellulose, 35S labeled proteins were visualized by autoradiography with film. For quantitation, the membrane was exposed to a phosphorimager screen and the resulting image was quantified in ImageJ. Differences in 35S incorporation were analyzed for statistical VEGF significance by one way ANOVA with Tukeys post test using the Prism software package. In vivo drug treatment and Western blotting. Drugs were prepared in 100 ll of vehicle containing 20% DMSO, 40% PEG 400, and 40% saline. Six wk old male C57BL/6 mice were fasted overnight prior to drug treatment. PP242, rapamycin, or vehicle alone was injected IP. After 30 min for the rapamycin treated mouse or 10 min for the PP242 and vehicle treated mice, 250 mU of insulin in 100 ll of saline was injected IP.

15 min after the insulin injection, the mice were killed by CO2 asphyxiation followed by cervical dislocation. Tissues were harvested and frozen on liquid nitrogen in 200 ll of cap lysis buffer. The frozen tissue was thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue homogenizer. Protein kinase inhibitor library for screening concentration of the cleared lysate was measured by Bradford assay and 5?10 lg of protein was analyzed by Western blot as described above. Cell proliferation assay. Wild type and SIN1_/_ MEFs were plated in 96 well plates at approximately 30% confluence and left overnight to adhere. The following day cells were treated with PP242, rapamycin, or vehicle.

After 72 h of treatment, 10 ll of 440 lM resazurin small molecule library sodium salt was added to each well, and after 18 h, the florescence intensity in each well was measured using a top reading florescent plate reader with excitation at 530 nm and emission at 590 nm. Supporting Information Figure S1. PP242 Inhibits Akt Phosphorylation over the Course of 1 h L6 myotubes were pre treated with PP242 or DMSO for 30 min and stimulated with insulin for the indicated times prior to lysis and analysis by Western blotting. Found at doi:10. 1371/journal. pbio. 1000038. sg001. Figure S2. Additional Analysis of 4EBP1 Phosphorylation 4EBP1 phosphorylation at T70 is not inhibited by either PP242 or rapamycin. L6 myotube lysates from Figure 6A were analyzed by Western blotting. 4EBP1 phosphorylation is inhibited by PP242 with similar potency in SIN1_/_ and wild type MEFs.

Western blotting from Figure 6B is shown with shorter exposures of p4EBP1. Found at doi:10. 1371/journal. pbio. 1000038. sg002. Figure S3.