Depletion of Smurf2 leads to a decline in NEDD9 protein levels. (A) Depletion of Smurf2 by siRNA (siSm2) leads to decreased NEDD9 protein levels in HeLa and CN34 cells. (B) Smurf2 does not affect NEDD9 transcript level. RNA samples from HeLa cells transfected with siSmurf2 were analyzed by RT-PCR for the indicated transcripts. (C) Forced expression of an siRNA-resistant Smurf2 mutant (Sm2siR) rescues NEDD9 levels in HeLa cells transfected with siSmurf2. D) NEDD9 depletion does not affect Smurf2 levels. For all samples, protein from HeLa cells was harvested 48 hours after transfection, 30 μg total protein was loaded onto gel. For siRNA transfections, cells were reverse transfected with 50 nM siRNA (siSmurf2#1, siNEDD9 Smartpool). RT-PCR was performed on cells treated as described above. RNA was extracted as described in Methods. For siRNA and DNA transfection, cells were forward transfected with 50 nM siRNA and 1 μg DNA of either no plasmid, CMV empty vector, FLAG-Smurf2si-resistant mutant.