Figure 3.

Effect of NRAS knockdown on c-Met phosphorylation. (a) Knockdown of mutated N-Ras by RNA interference in three cell lines carrying the Q61R NRAS mutation leads to diminished levels of phospho-c-Met. Cells were treated overnight with 100 nM siRNA specific for the NRAS mutation. Total cellular protein was prepared 72 hours after transfection, following a 7 minute exposure to HGF 100 ng/ml, and examined for N-Ras, phospho-c-Met, total c-Met, and actin by western blotting. (b) Knockdown of wild type N-Ras in cells lacking an NRAS mutation has minimal effect on c-Met phosphorylation. Experiments were performed in an identical manner to those described in (a) with the exception that the three cell lines carry wild type NRAS and the siRNA was therefore specific for the wild type message. (c) Densitometry results for phospho-c-Met (normalized to levels of total c-Met) in cells treated with non-targeting siRNA vs. NRAS-specific siRNA are shown. Quantitation of the above-described experimental results was performed with Image J software (NIH) using the ‘gelplot1’ Macro software. Declines in levels of phospho-c-Met in cells treated with non-targeting siRNA vs. NRAS-specific siRNA differed significantly between cell lines carrying mutant NRAS and those with wild type NRAS (p< 0.001). Unt, untreated; NT, non-targeting.