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. 2000 Dec 19;97(26):14200–14205. doi: 10.1073/pnas.97.26.14200

Figure 5.

Figure 5

A stable complex of hTFIIIB50 and associated factors of 54, 48, 42, and 40 kDa is required for transcription of the 7SK gene. (A) SDS/4–20% PAGE of 10 μl M2 eluates purified from nontransfected HeLa cells (lane 1) or HeLa cells that stably express hTFIIIB50 (lane 2). Construction of a HeLa cell line stably expressing fHA-tagged hTFIIIB50, as well as purification procedures on M2 agarose, were essentially as described (13). Purification of the hTFIIIB50 complex was performed in the presence of 500 mM KCl in BC buffer containing 0.05% NP-40. SDS/PAGE and silver stain were described (18). (B) Western blot with anti-hTFIIIB50 antibodies. Ten microliters M2 eluates purified from nontransfected HeLa cells (lane 1) or HeLa cells that stably express hTFIIIB50 (lane 2) was separated by SDS/5–20% PAGE and transferred onto nitrocellulose. SDS/PAGE and Western blot procedures were described (8). (C) Transcription of the 7SK gene. Transcription reactions were reconstituted with 1.5 μl cEDF 1 M, 24 ng rhTFIIIB150, and 40 ng rhTBP. The following fractions were added: lane 1, none; lanes 2 and 3, 10 and 20 μl cEDF 0.2, respectively; lanes 4–8, 20 μl flow-through (FT) of an anti-hTFIIIB50 antibody column onto which a cEDF 0.2 fraction had been loaded; and lanes 5–8, 5, 10, 20, and 40 ng of Ni-NTA agarose-purified rhTFIIIB50, respectively. Ni-NTA agarose-purified rhTFIIIB50 is shown in Fig. 2C. Transcription reactions were performed essentially as described (13). The film was heavily overexposed to analyze whether rhTFIIIB50 shows any residual activity in this assay system. (D) Transcription of the 7SK gene. Transcription reactions were reconstituted with 1.5 μl cEDF 1 M, 24 ng rhTFIIIB150, and 40 ng rhTBP. The following fractions were added to the reactions: lane 1, none; lanes 2–5, 2.5, 5, 10, and 20 μl cEDF 0.2, respectively; and lanes 6–10, 20 μl flow-through of an anti-hTFIIIB50 antibody column onto which a cEDF 0.2 fraction had been loaded. Reactions in lanes 7 and 8 or 9 and 10 contained in addition 10 or 20 μl M2 eluate, respectively, purified from HeLa cells that stably express fHA-hTFIIIB50 (lanes 7 and 8) or from control HeLa cells (lanes 9 and 10).