Inhibition of B-Raf by siRNA lowers VEGF-A expression in target cells. Target cells were untransfected or transfected either with ds siRNA or (NS)siRNA controls. (A) After 0, 12, 24, and 48 hours after transfection, total RNA was isolated from the cells and subjected to Northern blotting as per standard protocols to monitor B-Raf and β-actin mRNA. (B) Western blot analysis of phospho ERK1/2, total ERK1/2, and β-actin expression in the above cells was performed at 24 hours after transfection. (C) An ELISA was performed to quantify levels of VEGF-A present in the culture supernatant of various target cells. Each reaction was done in triplicate, and each point represents the average ± SD of 3 experiments.