B

1
DNAPK siRNA
A
2 siRNA
Control
C
ii)
120
i)
B
AA8 control
100
CHOV3 DNAPK-/-
80
35S-Met Incorporation
35S-Met Incorporation
60
40
20
0
Time (h)
Time (h)
A reduction in DNA-PKcs by RNAi also prevents
translational inhibition A) Western analysis of
DNA-PKcs levels in HeLa cells following
transfection with control siRNA (C2) or a siRNA
complementary to DNA-PKcs mRNA. There is a
reduction to approximately 50 of the expressed
protein in the presence of the siRNA. B) Protein
synthesis rates were determined by liquid
scintillation counting of newly incorporated
35S Methionine in mock treated cells and UVB
irradiated cells (275 J/m2). The data show that
in the cells that have reduced expression of
DNA-PKcs by RNAi (i) or in knockout mice (ii)
there is a reduction in the degree of
translational shut down following UV exposure.
Control GM00200
120
Seckle ATR-/-l GM18366
100
80
35S-Met Incorporation
60
40
20
0
0
4
Time (h)
C) Protein synthesis rates were determined by
liquid scintillation counting of newly
incorporated 35S Methionine in mock treated
cells and UVB irradiated cells (275 J/m2). The
data show that in the seckle cells that have
reduced expression of ATR (Stokes et al 2007)
there is a small reduction (15) in the degree of
translational shut down following UV exposure.
Supplementary 5