Figure 5

PI3K and Raf/MEK/Erk signaling in RoSH2 cells. a) Effects of PI3 K/Akt inhibitors. Undifferentiated RoSH2 cells were plated and cultured for 24 hours, then serum-starved for 20 hours before culturing in media with 50 μM LY294002 dissolved in DMSO, 100 nM wortmannin or 0.1% (v/v) DMSO for 15 min and then challenged with 100 nM insulin for 20 minutes. Cell lysates were prepared and assayed by western blot analysis; b) Raf/MEK/Erk signaling during differentiation. At 0, 6, 24 and 48 hours after plating RoSH2 cells on matrigel, cell lysates were prepared and analyzed by western blot assay for phosphorylated ERK1/2.. c) Effects of inhibiting Raf/MEK/Erk signalingRoSH2 cells were treated with 10 μM or 50 μM Ras inhibitor FTase inhibitor III, (FTI) and MEK inhibitor PD98059 for three hours and cell lysates were analyzed by western blot assays.