ces in the interaction of p85 with p110α vs. p110β. The exact nature of these differences and their consequences for PI3K function remain to be determined. Materials and Methods Plasmid Construction. LY2228820 The construction of the pBSFI vector encoding p110αH1047R, p110β, p110γ, and p110δ has been described previously. To facilitate cloning, the SfiI site in WT p85 was destroyed by point mutation.
The p85 mutant constructs were generated by using the QuikChange site-directed mutagenesis kit and the following primers: G376R : 5ATTATACTCTTACACTAAGGAAAGGGAGAAATAACAAATTAATCAAAATATTTC 3? G376R : 5?GAAATATTTTGATTAATTTGTTATTTCTCCCTTTCCTTAGTGTAAGAGTATAATC 3? E439del : 5?CAAATACCAACAGGATCAAGTTGTCAAAGATAATATTGAAGCTGTAGGG 3? E439del : 5?CCCTACAGCTTCAATATTATCTTTGACAACTTGATCCTGTTGGTATTTG Trichostatin A 3? KS459delN : 5?GAATATAACACTCAGTTTCAAGAAAATCGAGAATATGATAGATTATATG 3? KS459delN : 5?CATATAATCTATCATATTCTCGATTTTCTTGAAACTGAGTGTTATATTC 3?DKRMNS560del : 5?GGCAGCTGAGTATCGAGAAATCATTAAACCAGACCTTATCCAGCTGAG 3? DKRMNS560del : 5?CTCAGCTGGATAAGGTCTGGTTTAATGATTTCTCGATACTCAGCTGCC 3? D560Y : 5?GAAGCAGGCAGCTGAGTATCGAGAAATTTACAAACGTATGAACAGCATTAAACC 3? D560Y : 5?GGTTTAATGCTGTTCATACGTTTGTAAATTTCTCGATACTCAGCTGCCTGCTTC 3? N564K : 5?GTATCGAGAAATTGACAAACGTATGAAGAGCATTAAACCAGACCTTATCCAGCTG 3? N564K : 5?CAGCTGGATAAGGTCTGGTTTAATGCTCTTCATACGTTTGTCAATTTCTCGATAC 3? R574fs : 5?GCATTAAACCAGACCTTATCCAGCTGAAAGACGAGAGACCAATACTTG 3? R574fs : 5?CAAGTATTGGTCTCTCGTCTTTCAGCTGGATAAGGTCTGGTTTAATGC 3? T576del : 5?CCAGACCTTATCCAGCTGAGAAAGAGAGACCAATACTTGATGTGGTTG 3? T576del : 5?CAACCACATCAAGTATTGGTCTCTCTTTCTCAGCTGGATAAGGTCTGG 3? W583-del : 5?GAAAGACGAGAGACCAATACTTGATGTTGACTCAAAAAGGTGTTCGG 3? W583del : 5?CCGAACACCTTTTTGAGTCAACATCAAGTATTGGTCTCTCGTCTTTC 3? K379E : 5?GGAAAGGGGGAAATAACGAATTAATCAAAATATTTCATC 3? K379E : 5?GATGAAATATTTTGATTAATTCGTTATTTCCCCCTTCC 3? The mutated genes were subsequently cloned as SfiI DNA fragments into the avian retrovirus vector RCAS.
Sfi. To examine the binding of p85 mutants with p110 isoforms, the mutated p85 genes were FLAG-tagged and cloned into the pCAGGs vector by standard PCR and cloning. All clones were confirmed by sequencing. Cell Culture and Transfection. Fertilized chicken eggs were obtained from Charles River Breeding Laboratories. Primary CEF were prepared and cultured as described previously. For transfection, cells were plated at 80% confluence in F-10 containing 5.
8% iron-supplemented FCS and 1% L-glutamine�penicillin�streptomycin solution. On the following day, CEF were transfected with the RCAS vectors using the dimethyl sulfoxide/Polybrene method. After two passages in the presence of serum, the cells were harvested for further analysis. HEK 293-T cells were cultured in DMEM supplemented with 10% FCS and 1% L-glutamine�penicillin�streptomycin solution. Transfections were carried using lipofectamine-PLUS according to the manufacturers protocol. HEK293T cells in MP6 plates at 70% confluency were washed once with Opti-MEM medium and incubated in 0.8 mL of Opti-MEM. A total of 1 μg of plasmid DNA was mixed with 0.1 mL of Opti-MEM and 2 μL of Lipofectamine PLUS for 15 min at room temperature. Opti-MEM with 6 μL of Lipofectamine was added to the DNA-PLUS mixture and incubated for 15 min at room temperature.
The mixture of DNA, PLUS, and Lipofectamine was added to the cells and incubated overnight. The second day, the medium was changed to DMEM containing 10% FCS and 1% Lglutamine�penicillin�streptomycin solution. Forty hours after transfection, the cells were collected, lysed, and analyzed for specific proteins. Focus Assay. Focus assays with infectious retroviral vectors were performed as previous