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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

PI3K

PI3K Substrate Competition Assay Cells were plated as described above in the PI3K Activity Assay section. Cells were treated with 10% FBS for 30 min following 24 h of serum starvation. Cells were not treated with retinol prior to lysis. Cells were lysed with cell lysis buffer as described in the PI3K Activity Assay section, above. PI3K proteins were immunoprecipitated by incubation of 400 μg of whole cell protein lysate with 2 μg of p110 antibody overnight at 4°C. Following incubation, 25 μl of sepharose 4A beads (50 mg/ml) were added for 2 h at 4°C. The beads were washed twice with 1% NP-40 and once with each of the following; PBS, LT, TEN, and TGN. The washed beads were resuspended in 50 μl TGN buffer. Ten μl samples were subjected to western blot analysis for p110 levels to serve as an internal loading control, as described above. Immunoprecipitated PI3K was incubated with 10 μg PI, retinol vehicle (ethanol) or increasing concentrations of retinol (0, 10, 20, 50, and 100 μg; corresponding to 0, 0.6, 1.2, 2.9, and 5.8 μM retinol, respectively) for 10 min at room temperature prior to the addition of 10 μCi of [γ- 32 P] ATP and 20 mM MgCl2 for 20 min at room temperature. In addition, immunoprecipitated PI3K was incubated with 10 μg retinol and increasing concentrations of PI (10, 20, and 50 μg). The total volume of ethanol vehicle was the same in all samples, regardless of retinol concentration. Lipid extraction and TLC were performed as described above in the PI3K Activity Assay section. 75

Invasion Assays Cells were serum starved for 24 h before seeding at a density of 1 x 10 5 cells/well onto Boyden chambers coated with Matrigel. The upper portion of the chambers contained 0 (ethanol vehicle control) or 10 μM retinol and 0, 10 or 50 μM of the PI3K inhibitor, LY294002. DMEM containing 10% FBS was added to the lower portion of each chamber to serve as a chemoattractant. After 24 h, all cells were removed from the upper chamber using a cotton swab. HCT-116 cells that had migrated through the membrane were stained with crystal violet. SW620 cells that had migrated through the membrane were fixed in methanol prior to staining with propidium iodide. The bottom of the membrane was examined microscopically and the number of stained cells present in ten random fields of view counted to determine cell invasion as described (96). Computational Methods The Hyperchem 7.5 modeling environment was used to perform a series of calculations on retinol, wortmannin and a fragment of PI. To decrease the computational complexity the native PI structure was fragmented by decreasing both of the ester chains to a single methyl group. This model compound should remain representative of the larger structure as the activity of PI at PI3K is focused near the cyclohexyl and phosphate groups and not at the non-polar side chains, which instead interact with the cell membrane (147). Structure pre-optimization was conducted using semi-empirical quantum mechanics with a MNDO/d parameterization. Resulting structures were then carried forward to a more complex ab initio optimization using a 76

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