Figure 5

Inhibition of ERK1/2 and SFK attenuates SK1 expression and enzymatic activity and VEGF expression. MDAMB-231 cells were starved overnight in serum-free media and pre-treated with MEK1/2 inhibitor U0126 (10 μM) and/or SFK inhibitor SU6656 (10 μM) for 1 h followed by stimulation with 1000 ng/ml leptin for 6 h. (A) Cell lysates were separated on 10% SDS-PAGE gel and probed for phosphorylation of STAT3, SFK and ERK1/2. Blots are representative of three independent experiments. SK1 (B) and VEGF (D) expression and SK1 activity (C) were measured in cell lysates containing equal amounts of mRNA and protein. SK1 activity was measured by radiolabelling of sphingosine. For qRT-PCR, SK1 and VEGF were normalised against housekeeping genes (GAPDH, YWHAZ and UBC) and analysed using qBase software. Columns, mean of three independent experiments performed in triplicate; bars, SEM. (*, P <0.05; **, P <0.01; §, P <0.001; NS, not significant, P >0.05).