FIG. 8.

Inhibition of EGFR1 internalization mimics EGFRvIII-mediated effects on TBP promoter induction. (A) Decreased clathrin expression in U87-EGFR1 cells alters TBP promoter induction through the AP-1 site. Cells were transfected with either mm or clathrin-specific siRNAs and cotransfected with TBP-luciferase and CMV-β-galactosidase promoter-reporter constructs. Cell lysates were analyzed for luciferase and β-galactosidase activities. Luciferase activity levels were normalized to β-galactosidase activity, and changes were calculated based on untreated cells transfected with mm siRNA. (B) Decreased clathrin expression does not affect EGFRvIII-mediated induction of TBP promoter activity through the AP-1 site. Experiments were conducted as described in panel A using U87-EGFRvIII cells. (C) Decreased clathrin expression in U87-EGFR1 cells increases the recruitment of c-jun and c-fos while decreasing the recruitment of Elk-1 to the TBP promoter. ChIP assays were performed on cells transfected with either mm (−) or clathrin-specific siRNA using Elk-1, c-jun, and c-fos antibodies. qPCR was performed to quantify the amplified DNA using the primer sets as described for Fig. 5. The n-fold change in TBP occupancy was calculated based on untreated mm control.