ly described. CEF were transfected with the appropriate RCAS constructs using the dimethyl sulfoxide/Polybrene method and overlayed with nutrient agar every other day for 2 to 3 wk until focus formation was observed. The plates were stained with crystal violet, and foci of transformed cells were counted. To examine inhibition of focus formation by different NVP-BEP800 VER-82576 compounds, 10 μM LY294002, 100 nM NVP-BEZ-235, 2 nM rapamycin, 5 μM ZK-93, 250 nM TGX221, 5 μM IC87114, or 5 μM AS604850 were added to the nutrient agar in every overlay. Cell Proliferation. After transfection CEF were split into proliferation assay media containing F-10 supplemented with 2% FCS and 1% chicken serum. At the second split cells were seeded into a 96-well plate at 4,000 cells per well.
On days 1 to 5 after seeding, CEF were incubated with 10 μg/mL Resazurin-Na in proliferation assay media for 4 h at 37 °C. Fluorescence was determined at an excitation wavelength VX-680 of 560 nm and an emission of 590 nm. Western Blot and Immunoprecipitation. Western blotting was performed as previously described , with minor modifications. Cells were lysed in modified Nonidet P-40 lysis buffer. After centrifugation for 10 min at 18,000 × g at 4 °C, the protein concentration of the supernatant was determined. For the examination of inhibitor signaling, the cells were treated with 250 nM TGX-221 or 5 μM IC87114 for 2 h in the serum-containing condition ahead of collecting. For immunoprecipitation, cell lysates containing 40 μg of protein were incubated with anti-FLAG M2 Agarose overnight at 4 °C.
Agarose beads were washed four times with lysis buffer and heated to 95 °C before sepa-Fig.7. Effect of the p110α-specific inhibitor A66 on mutant signaling. Signaling to Akt by KS459delN and DKRMNS560del was reduced by the inhibitor. Sun et al. PNAS | August 31, 2010 | vol.107 | no.35 | 15551 MEDICAL SCIENCES ration on an SDS/PAGE gel. After transfer to Immobilon P membranes these membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween-20 for 2 h at room temperature and then incubated with a dilution of 1:2,000 of anti-FLAG or 1:1,000 of anti-p110α or anti-p110βprimary antibody overnight. Membranes were washed three times in TBS-T and incubated with peroxidase-coupled goat anti-mouse or goat anti-rabbit antibody for 1 h in 5% BSA/TBS-T at room temperature.
The reactive bands were visualized by SuperSignal West Pico Chemiluminescent substrate. For Western blotting, cell lysates containing 10 μg of total protein were separated on SDS/PAGE gels and transferred to Immobilon P membranes. Membraneswere incubatedwith 1:1,000 dilutions of primary antibodies directed against p85, pAkt , Akt, p4E-BP, 4E-BP, and β-actin.Western blots were developed as described above. Anti-FLAG antibody was purchased from Sigma-Aldrich. Anti-p110α antibody , anti-p110 β antibody , anti-p85 antibody , anti-Akt , anti-phospho-Akt , anti-4E-BP1 , and anti-phospho-4E-BP1 antibodies were obtained from Cell Signaling Technology. Anti-p110δantibody was purchased from Santa Cruz Biotechnology. Isoform-Specific Inhibitors. The isoform-specific inhibitors for p110β, p110γ,and p110δ have been described in a previous publication.
The specific inhibitor A66 for p110α was synthesized and characterized as described previously. ACKNOWLEDGMENTS. We thank Lynn Ueno for expert technical support. ZK-93 was a kind gift from Kevan Shokat. We thank Yen Hoang Le Nguyen for stimulating and insightful discussions. This work was supported by grants from the National Cancer Institute. This is Manuscript 20600 of The Scripps Research Institute.1. Samuels Y, et al. High frequency of mutations of the PIK3CA gene in hu