Fig. 1.
Overexpression of GMF in mouse neuroblastoma N18 cells after transfection with GMF/adenovirus construct (GMF-V). Mouse N18 cells were plated into 24-well plates (1×105 cells per well) and grown in DMEM/F12 containing 5% fetal bovine serum (complete medium). Replication-defective human adenovirus vector containing a full length GMF cDNA (GMF-V) or cytoplasmic lacZ cDNA (LacZ) at 20 MOI (multiplicity of infectivity) were added to cells in serum-free and antibiotic free DMEM/F12 medium for 4 h. Cells were rinsed and allowed to grow in fresh complete medium for another 24 h. Mock-transfected controls were processed identically in the absence of virus. Cell lysates (20 μg protein per lane) were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting. The blots were probed with anti-GMF (G2-09) antibody and anti-β-actin antibody. Actin served as internal marker showing equal sample loading. For siRNA mediated down-regulation of GMF expression in N18, cells were transfected with duplex siRNA targeted against GMF (GsiRNA 10 nM), or with a control scrambled sequence (CsiRNA 10 nM). Cells were transfected with siRNA 4 h prior to adenovirus transfections. Lane 1, mock infection; lane 2, lacZ; lane 3, GMF-V; lane 4, CsiRNA; and lane 5, GsiRNA. (A) Western blot. (B) RT-PCR. The data shown are representative of at least three experiments.