Effect of hRFVT-2 predicted and clinically relevant mutations on level of expression of hRFVT-2 mRNA and protein in U87 cells. A&B) Total RNA was isolated from WT, predicted and the clinically relevant indicated mutant constructs stably expressing U87 cells and quantitative real-time PCR was performed. Data (means ± SE) are from at least 3 independent experiments, normalized relative to β-actin. C&D) Western blot was performed using equal amounts (60 μg) of total protein isolated from the wild-type, predicted and clinically relevant indicated mutant constructs expressing cells cell-lysates. The blot was probed with the anti-GFP monoclonal and β-actin polyclonal primary antibodies. Data (means ± SE) from at least 3 independent sets of experiment are expressed in arbitrary units. Inset is the image of a representative gel. *P < 0.01, **P < 0.02, ***P < 0.05.