Figure 4

KU0063794 inhibits global protein translation, while increasing translation of the MGMT protein. Cells were incubated in the absence or presence of KU0063794. A. Cell lysates were prepared, polysomes fractionated using sucrose density gradients. The UV profile from this analysis is shown here and the fractionation of 40S, 560S, 80S ribosomes and polysomes is indicated. mRNA from pooled fractions was subsequently extracted as described in the Materials and Methods. For each gradient, qRT-PCR was used to determine levels of GAPDH and MGMT mRNA distribution across each fraction (free represents RNA and associated material at the top of the gradient and subunits show 40S/60S/80S-bound RNA), with the total mRNA in each gradient set at 100%. Cells were incubated for 24 hours in the absence (B) or presence of KU0063794 (C).