Silencing of InsR attenuated cellular accumulation of FN in MES-13 mesangial cells. (A) Immunoblotting with anti-FN antibody was performed on clones of InsR silenced cells and SC transfected cells. An image of gel stained after transfer was shown as a loading monitor (middle panel). Cellular FN levels were evaluated by densitometric analysis. Data are means ± SEM from four independent experiments. *, P<0.01 versus SC transfected cells (lower panel). (B) Semi-quantitative analysis of FN mRNA levels in InsR silenced and SC transfected MES-13 cells. Total RNA isolated from quiescent cells was subject to RT–PCR using FN and GAPDH (internal control) specific primers with the indicated number of amplification cycles. One representative image from four independent experiments is shown (upper panel). FN transcription levels were evaluated by densitometric analysis. Data are means ± SEM from four independent experiments.