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. 1998 Mar 3;95(5):2192–2197. doi: 10.1073/pnas.95.5.2192

Figure 2.

Figure 2

PC4 does not interfere with formation of TBP-mediated PICs. (A) For EMSA, an end-labeled Ad ML oligonucleotide (−40 to +10) probe was incubated with the indicated combinations of factors: TFIIB (B, 20 ng, lanes 2–4, 7–9, 12–14, and 17–19); TFIIF (F, 25 ng, lanes 3, 4, 8, 9, 13, 14, 18, and 19); pol II (II, lanes 4, 5, 9, 10, 14, 15, and 19). Reactions were carried out in the absence (lanes 1–5 and 11–15) or presence (lanes 6–10 and 16–19) of native TFIIA (A). PC4 (75 ng) was added in lanes 11–19. All reactions contained TBP (0.5 ng). In the absence of TFIIB and TFIIF, pol II yields a nonspecific band (lanes 5, 10, and 15). Reactions were incubated at 30°C for 30 min and resolved by native PAGE. Free probe has been excised from the autoradiograms. (B) EMSA reactions were performed as in A with either wild-type (lanes 1–5) or N-terminal truncated TFIIB (ΔB, lanes 6–10). TFIIA was added to reactions 1 and 6; pol II and TFIIF (II/F) to reactions 4, 5, 9, and 10; and PC4 to reactions 3, 5, 8, and 10. All reactions contained TBP. (C) EMSA reactions contained TBP and PC4 and, as indicated, TFIIB (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18) and pol II plus TFIIF (lanes 3, 6, 9, 12, 15, and 18). Preimmune (pre, lanes 4–6), anti-TBP (lanes 7–9), anti-TFIIB (lanes 10–12), anti-CTD (pol II, lanes 13–15), and anti-PC4 antisera were added after 10 min of incubation at 30°C.