Figure 4

Signalling pathway activation analysis. A) Quantification of active Ras present in ASP13 and CYS12 cells. ASP13 transfectants showed more Ras activation (P = 0.026). B) Analysis of p-ERKs, p-Akt and Tubulin (as a loading control) from ASP13 and CYS12 cells treated or not with U0126 (specific inhibitor of MEK and in consequence of p-ERK) and LY294002 (specific inhibitor of PI3K and in consequence of p-Akt) is depicted. p-ERKs levels were higher in ASP13 transfectants than CYS12, while no differences were observed in p-Akt levels. Representative western blots are shown. C) mRNA VEGF-A levels as 2^-ΔCT; ASP13 Control 0.40 ± 0.07; +LY294002 0.49 ± 0.1, +U0126 0.12 ± 0.04; CYS12 Control 0.08 ± 0.009, +LY294002 0.05 ±0.02, +U0126 0.046 ± 0.02. Treatment with U0126 MEK inhibitor but not with PI3K inhibitor LY294002 resulted in a reduction of mRNA VEGF-A levels in ASP13 transfectants (P = 0.031). D) Sp1 transcription factor levels were not different in ASP13 and CYS12 cells. Levels of Sp1 and Tubulin (as a loading control) were assayed in extracts from NIH3T3 cells (N), different ASP 13 clones (4, 5, 6 and 7) and different CYS 12 clones (1, 2 and 3).