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. 2015 Sep 17;290(44):26832–26845. doi: 10.1074/jbc.M115.688317

FIGURE 1.

FIGURE 1.

The NF complexes possess a direct transcriptional activity as coactivators. A, purification scheme of the TREF activities from HeLa cell nuclear extracts (NE). The numbers indicate the molar concentration of KCl in the elution buffer. In a silver-stained SDS-polyacrylamide gel of the purified TREFγ, the positions of NF45 and NF90 are indicated on the right, and the arrowhead indicates the position of NF110. B, structures of NF45, NF90, and NF110. The number of amino acid residues is shown at the C terminus of each NF protein. RGG, DZF, NES, NLS, and dsRBM indicate arginine-glycine-glycine rich, dimerization zinc finger, nuclear export signal, nuclear localization signal, and double-stranded RNA-binding motif, respectively. The regions of the NF proteins used for immunizing rabbits are indicated by a black bar under the schematic structures of NF45, NF90, and NF110. Because 353–706 of NF90 is identical to 353–706 of NF110, the antibody raised against this region is designated as anti-NF90/NF110. C, NF45, NF90, and NF110 were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). The antibodies were tested for their reactivity with NF45, NF90, and NF110. Anti-NF45 and anti-NF110 detected NF45 and NF110, respectively (WB). As expected, anti-NF90/NF110 detected both NF90 and NF110. D, the immunoblot with anti-NF110 (WB) revealed that the TREFγ fraction contained a small amount of NF110 as indicated by an arrowhead (left panel, silver staining). E, SDS-PAGE analysis of purified recombinant NF45-NF90 (lane 1) and NF45-NF110 (lane 2). F, comparison of the transcriptional activities of TREFγ and NF45-NF90 using in vitro transcription of pfMC2AT. Activators indicates 20 ng of SRF, 10 ng of Elk-1, 20 ng of CREB, 20 ng of ATF1, and 200 ng of PC4 (lanes 2, 4, and 6). Semi-quantitative immunoblots with anti-NF45 antibody were used to estimate the amounts of the NF complexes in the TREFγ and recombinant NF45-NF90, and the reactions contained 6.7 μl of TREFγ (lanes 3 and 4) and 90 ng of NF45-NF90 (lanes 5 and 6), respectively. The arrow indicates the position of the transcript, and the relative levels of the transcripts are indicated below each lane. G, in vitro transcription reactions were performed as in F in the presence of the indicated amounts of NF45-NF90 and NF45-NF110. The relative transcription levels are indicated below each lane.