Figure 4

eIF6 over-expression increased polysome loading of cdc42 mRNA. The polysomal profiles of A2780/eIF6 and control cells were analysed by density gradient centrifugation. The sucrose gradient fractions were pooled together on the basis of the presence/absence of ribosomes, detected by ethidium bromide staining on agarose gels (upper panel). The total RNA of each polyribosomal fraction was extracted. Successively, cdc42 mRNA was measured in both fractions by RT-qPCR (bottom panel). The amount of cdc42 mRNA in the polysomal fractions was normalized using rRNA as the standard, while for ribosome-free fractions we used GAPDH mRNA levels. We also analysed GAPDH mRNA levels in the polysomal fractions normalizing with respect rRNA levels. The mean value is representative of three independent experiments with a P-value < 0.05 (**) and < 0.01 (*) respectively, calculated with the t-test.