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. 2000 Jun 13;97(13):7184–7189. doi: 10.1073/pnas.140202297

Figure 3.

Figure 3

(A) RNAP II purified from HeLa nuclear pellet restores HIV-1 enhancer-dependent transcription activation in NE[ΔNC2β]. Standard transcription reactions contained 25 μg HeLa NE proteins and were analyzed as described in the legend to Fig. 1. (B) Purified recombinant 6His:tagged NC2 (6His:rNC2) after SDS/PAGE and Coomassie staining. (C) Addition of 6His:rNC2 in amounts comparable to those of NC2 present in untreated HeLa NE does not affect transcription levels in NE[ΔNC2β]. (D) 6His:rNC2 selectively interacts with the CTD-phosphorylated IIO form present in RNAP II purified from HeLa nuclear pellet. RNAP II immunoprecipitated with anti-NC2β antibodies after preincubation in the absence (lanes 5 and 6) or presence (lanes 3 and 4) of 6His:rNC2. Twenty percent of input proteins, 50% of the unbound fractions (U), and 100% of the immunoprecipitates (B) were subjected to SDS/PAGE and immunoblot analysis. The large RPB1 subunit of RNAP II was analyzed by using antibodies H5 and N-20 described in the legend to Fig. 2.