Figure 5.

(A, panel i) Cell lysates from M059K or M059J cells were prepared separated by SDS-PAGE and immunoblotted for DNA-PKcs. As expected, no DNA-PKcs was detected in M059J cells. (panel ii) M059K or M059J cells were exposed to UVB light, and protein synthesis rates were determined by liquid scintillation counting of newly incorporated [³⁵S]-methionine. Measurements are the mean of three independent experiments normalized to that of the nonirradiated cells. Error bars represent one standard deviation from the mean. (B) Cell lysates from M059K (panel i) orM059J (panel ii) cells were exposed to UVB light and immunoblotted for eIF2α, GCN2, and PERK. The data show that there in no change in the degree of phosphorylation of eIF2α or GCN2 in cells that lack DNA-PKcs. (C) M059K (panel i) orM059J (panel ii) cells were exposed to UVB light and, after 4 h, cell lysates were prepared and fractionated using sucrose density gradient analysis on 10%–50% (w/v) sucrose gradients. (D) Cell lysates were generated from M059K (panel i) orM059J (panel ii) cells ± exposure to UVB light and applied to 10%–50% (w/v) sucrose gradients. mRNA was isolated from individual fractions, and Northern analysis was performed to determine the polysome/subpolysome distribution of DDB1, ERCC1, and ERCC5.