Stable transfection of shRNA against InsR in MES-13 mesangial cells. Cells (90% confluent) were stably transfected as described and serum-deprived for 16 hours. Immunoblotting with anti-InsRα antibody was performed on three clones of InsR silenced cells (clone 7, 11, 13) along with scrambled oligo (SC) transfected cells. An image of gel stained after transfer was shown as a loading monitor (middle panel). InsR expression levels were evaluated by densitometric analysis. Data are means ± SEM from four independent experiments. *, P<0.01 versus SC transfected cells, WT, wild type.