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Fig. 3. Association detected by co-purification. (A) Immunoblot (using antibody directed against N terminus of the largest subunit) showing form IIA and IIO content in fractions released from HeLa cells treated successively with a hypotonic buffer (`cytosol', lane 1), and 0.035, 0.14, and 0.65 M (NH4)2SO4 (`nuclear', lanes 2-4) and RNase A (`RNase', lane 5). After RNase treatment the fraction contains an especially hyper-phosphorylated form (IIO*). Polymerizing activities (pmol UTP incorporated/min/µg protein) sensitive to {alpha}-amanitin were: 2.7 [polymerase purified by a conventional procedure (Maldonado et al., 1996)], 0.3 (cytosol), 0.4 (0.035 M), 0.2 (0.14 M), 0.3 (0.65 M), 3.5 (sub-nuclear) and 0.1 (negative control lacking DNA). (B) Immunoblots of the RNase-treated fraction before (`input' with 1x and 1/3x loadings in lanes 1 and 2) and after immunopurification using either antibodies directed against the N terminus of the largest catalytic subunit (`anti-pol', lane 3) or normal mouse serum (lane 4). eIF4E, S6, UPF1 and UPF2 co-immunopurify with the forms IIA and IIO (pol) and the CTDP; in contrast, XPB, cdk7, and cdk8 do not.





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