Figure 6

Effects of down-regulation of RasGRP3 on the Ras signaling pathway I. RasGRP3 is involved in IGF-I and EGF (A) dependent Akt, ERK and ERα phosphorilation. RasGRP3 knockdown cell lines created from MCF7 and T-47D cells were treated with or without IGF-I (100 pg/ml) and EGF (100 pg/ml) as indicated. Akt and phosphorylated Akt, ERK and phosphorylated ERK, ERα and phosphorylated ERα were detected by immunoblotting of cell lysates. Levels of total Akt, ERK and ERα were used as control. All results were representative of 2 independent experiments. (B) Q-PCR analyses of ERα-regulated genes, namely progesterone receptor (PGR), cathepsin D (CTSD), cytochrome C (CYCS) and loricrin (LOR). GAPDH was used as an internal control. The results are representative of three independent experiments. Values represent the mean ± SEM. In case of T-47D cells loricrin expression *indicates significant difference compared to non-transfected control cells, while ^#indicates significant difference compared to scrambled control cells.