Figure 1
Differential EGFR levels and ERK phosphorylation in RCC cells. (A) Aliquots (10 μg) of cell lysates were analysed by Western blot for levels of EGFR protein. PRC3, MPR6 and WT8 are derived from 786-O cells by transfection of empty vector (PRC3) or independent wild-type VHL expression constructs (MPR6 and WT8). Tubulin served as a loading control. (B) RCC cell lines WT8VHL-wt and PRC3VHL-mut were grown until 50% confluent, then treated for 2 h with DMSO (lanes 1 and 5), rapamycin (10 nM; lanes 2 and 6), Iressa (10 μ M; lanes 3 and 7) or both (lanes 4 and 8). Aliquots were analysed for the indicated total and phospho-proteins. (C) WT8VHL-wt and PRC3VHL-mut cells at 50% confluency were treated for 2 h with decreasing doses of Iressa (10–0.1 μ M) and analysed as above. (D) WT8VHL-wt cells at 50% confluency were treated for 20 h with 10 or 100 μ M CoCl2, or not, as indicated (Co:). For the last 2 h, selected cultures were treated with rapamycin (10 nM), Iressa (10 μ M) or both; control cells were treated with DMSO. Cells were washed twice with PBS, harvested and analysed with the indicated antibodies. One lane of PRC3VHL-mut lysate was included as a positive control for the HIF2α antibody (arrow). This lane was not analysed with the other four antibodies.
