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. 2004 May 10;101(20):7572–7577. doi: 10.1073/pnas.0401493101

Fig. 4.

Fig. 4.

DSIF can overcome FCP1-mediated capping inhibition. (A) Ternary +24 complexes alone (lane 1) or incubated with 0.6 ng of CE and GTP for 5 min at 30°C (lane 2). Ternary complexes were incubated with 60 ng of FCP1 for 30 min at 30°C, washed to remove FCP1, resuspended in TB60, and treated with CE and GTP in the absence (lane 3) or presence (lanes 4-6) of varying amounts of DSIF. As control, complexes not treated with FCP1 were incubated with DSIF and CE (lanes 7-8). (B and C) Capping reactions in A were subjected to 6% SDS/PAGE and analyzed by Western blot with GAL4-CTD polyclonal antibody, which recognizes both phosphorylated (IIO) and nonphosphorylated (IIA) Rpb1 and monoclonal antibody H14 to detect specifically Ser-5 phosphorylated CTD. (D) Quantitation of A. (E) Ternary +24 complexes were treated with FCP1 for 30 min, and treated and untreated complexes were incubated with 0.6 ng CE in the absence and presence of 30 ng of DSIF. Reactions were stopped at the indicated times, and the percentage of capped RNA was determined by SDS/PAGE and autoradiography.