Figure 6
Analysis of primary tumours. (A) Protein (10 μg) from 12 matched pairs of renal tumours (T) and normal kidney (N) were analysed with the indicated antibodies on two separate sets of blots. Equivalent gels were stained with Coomassie blue (CBB) for loading. The low protein content of tumour #10 indicates that relative Erk phosphorylation was even higher than the band intensity suggests. VHL mutational status is summarised by m=mutant; w=wild type. (B) In total, 30 μg of tumour and normal lysates were analysed for levels of EGFR, phospho-RPS6 and total RPS6. The four cell line lysates were treated with Iressa and rapamycin (I/R) for 2 h or not (−). Densitometric analysis for the left panel was normalised to the tumour with the lowest EGFR signal. Densitometry for the right panel utilised a shorter exposure to remain within the linear response range. Even the longest exposures failed to detect EGFR in T-12. The asterisk denotes a background band. (C) Direct comparisons of phospho-Erk and phospho-Akt levels in a subset of tumours and cell lines on the same filter. (D) WT8VHL-wt cultures starting at 70% confluency (time zero) were grown under standard culture conditions over 48 h and periodically sampled. Phospho-RPS6 levels were downregulated at 100% confluency.
