Figure 4.
Uncleavable TFIIA is more stable than cleavable TFIIA. (A) U2-OS cells transfected with plasmids expressing hTFIIAγ together with Myc-tagged hTFIIAαβ wt (lanes 2–6) or mutant G275A (lanes 7–11) were labelled for 1 h with [35S]Trans followed by a chase for 1, 2, 4, 8 and 24 h. Extracts from these cells were subjected to immunoprecipitation using the Myc antibody followed by SDS–PAGE and fluorography. (B) Quantitation of labelled proteins from (A) was performed by Phosphoimager. The results represent the average of three independent experiments. (C) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ wild-type or mutant G275A and treated with CHX were analysed by SDS–PAGE and immunoblotting using antibodies against TFIIAα and GFP. Plasmid expressing GFP was cotransfected as the internal control. (D) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ wild type or mutants as indicated and treated with CHX were analysed by SDS–PAGE and immunoblotting using antibodies against TFIIAα. (E) Quantitation of (D) was performed by Phosphoimager. The result represents the average of three independent experiments.