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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

immunoprecipitated by incubation of 400 μg of whole cell protein lysate with 4 μg of IRS-1 antibody (Upstate, #06-248, Lake Placid, NY) overnight at 4°C. The next day, 50 μl of protein A/G plus-agarose beads (Santa Cruz Biotechnology, #sc-2003, Santa Cruz, CA) were added to the lysate and incubated for 2 h at 4°C. PI3K activity was determined as described previously (145) with minor modification as follows. The beads were washed twice with 1% NP-40 and once with each of the following: PBS, LT (lithium Tris) buffer [0.5 M LiCl and 200 mM Tris-HCl (pH 7.5)], TEN (Tris, EDTA, sodium chloride) buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 100 mM NaCl), and TGN (Tris, EGTA, sodium chloride) buffer (20 mM Tris-HCl (pH 7.5), 1 mM EGTA, and 100 mM NaCl). The washed beads were resuspended in 40 μl TGN buffer. Immunoprecipitated PI3K was incubated with 10 μg phosphatidylinositol PI (Avanti Polar Lipid, Alabaster, AL) for 10 min at room temperature followed by incubation with 10 μCi of [γ- 32 P] ATP and 20 mM MgCl2 for 20 min at room temperature. The reaction was stopped with 100 μl 1N HCl. The samples were centrifuged for 1 min at 15,000 x g. Following centrifugation, the supernatant was extracted with a 1:1 solution of chloroform:methanol and the lower fraction spotted on TLC (thin layer chromatography) plates (Fisher Scientific Company, Houston TX). Before use, the TLC plate was treated with coating solution (40% methanol, 1.2 mM EDTA and 1% potassium oxalate) for 30 min and dried overnight. TLC separation buffer consisted of chloroform:methanol:30% ammonium hydroxide:H20 = 129:114:15:21. Dried TLC plates were exposed to film at –70°C for 3 to 5 d. 73

Western Blot Analysis Cells were plated at a density of 1 x 10 6 cells/100 mm dish. Cells were treated with 0, 1, and 10 μM retinol for 30, 60, 120 and 240 min after 24 h of serum starvation. Western blot analysis was performed as described previously (146). Protein (50 μg) was electrophoresed through 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were incubated with a 1:100 dilution of p110 antibody (Santa Cruz Biotechnology, #sc-8010, Santa Cruz, CA) in 5% BSA in TBST (Tris-buffered saline, Tween) overnight at 4°C or with a 1:100 dilution of p85 antibody (Santa Cruz Biotechnology, #sc-423, Santa Cruz, CA) in TBST overnight at 4°C. Immunoprecipitation of PI3K Cells were plated as described above in the PI3K Activity Assay section. Cells were treated with 0 and 10 μM retinol for 5, 10, 20, 30, 60, and 120 min after 24 h of serum starvation. Cells were lysed with cell lysis buffer as described in the PI3K Activity Assay section, above. PI3K protein was immunoprecipitated by incubation of 200 μg of whole cell protein lysate with 2 μg of p110 antibody overnight at 4°C. The following day, 25 μl of sepharose 4A beads (50 mg/ml) (Amersham Biosciences, Piscataway, NJ) were added to the lysate prior to incubation for 2 h at 4°C. The beads were washed twice with 1% NP-40 and once with PBS. Protein samples were subjected to western blot analysis as described above. 74

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