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Fig. 3. Expression of an inducible active form of PI3K in HaCaT keratinocytes. (A) The retroviral vector used for expression of the Myr-p110^*-mER fusion protein is shown. IRES, internal ribosomal entry site; iSH2 (p85), inter SH2 domain of the murine p85 protein; L, linker sequence; LTR, long terminal repeat; mER, cDNA encoding the mutated murine estrogen receptor ligand binding domain; mp110^*, murine p110 cDNA; Myr, myristoylation signal sequence; neo, neomycin-resistance gene. The position of the riboprobe p110^*-p used for RPA is indicated as a black line. (B) Western blotting with 30 µg total cellular protein for detection of endogenous p110 and overexpressed Myr-p110^*-mER protein in HaCaT cell clones stably transfected with a retroviral Myr-p110^*-mER construct. (C) Western blotting using 30 µg total cellular protein revealed increased amounts of phosphorylated Akt after 4-OHT-treatment of the stable cell clones 1 and 17. The amount of ß-actin in each lane served as a loading control. (D) As shown by western blot analysis of phosphorylated Akt, induction of PI3K signaling by 4-OHT was blocked by simultaneous addition of LY294002. Simultaneous detection of PCNA indicates that cell proliferation is not negatively influenced by LY294002 treatment during this time period. (E) Vector-transfected and Myr-p110^*-mER expressing keratinocytes were treated with 4-OHT (+) or solvent (-) as indicated. DNA replication was measured by [³H]thymidine incorporation. Untreated control was arbitrarily set as 1.0. Western blot analysis, which was carried out in parallel, verifies phosphorylation of Akt after 4-OHT-treatment in Myr-p110^*-mER cell clones. Error bars indicate s.e.m. Significance was determined with the Student's t-test (two-tailed). ^**P<0.01; ^***P<0.001.

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