Fig. 4. The dynamics of Akt activation in cultured human keratinocytes. (A) Adherent keratinocytes that had been starved for 24 hours were treated with 400 ng/ml recombinant Gas6 for 0-40 minutes. Western blots were probed with antibodies to phospho-Akt⁴⁷³ or total Akt. Erk2 was used as a loading control. (B) FOXN1ER transduced keratinocytes were stimulated with 4OHT (100 nM) for 0, 6, 12 or 24 hours and lysates were probed for phospho-Akt⁴⁷³, total Akt or Erk2. (C) Keratinocytes were held in suspension for 0 to 28 hours and total Akt protein was immunoprecipitated. The level of phosphorylated GSK-3ß substrate was analysed by western blotting as a measure of Akt kinase activity. Actin was used as a loading control. (D) Percentage of involucrin-positive cells induced by FOXN1 or suspension culture in the presence or absence of PI 3-kinase inhibitor LY294002 (LY) or Akt inhibitor. The means and standard errors are shown from three experiments.