Figure 3.
Evaluating the specific binding of the RNAP2 Ser2ph-mintbody to RNAP2 Ser2ph in living cells.(A and B) HeLa cells that stably express the RNAP2 Ser2ph-mintbody were treated with 1 µM flavopiridol, 5 µM triptolide, or vehicle (0.1% DMSO) for 2 h. High-resolution confocal images were acquired using a high-resolution confocal system. (A) Representative images at 0, 60, and 120 min. Scale bar, 5 µm. (B) Nuclear foci were selected using autothresholding, and the total area in the single nucleus was measured. The areas relative to time 0 were plotted with SEM (n = 10). The area of foci decreased by treatment with flavopiridol and triptolide. (C) FRAP. A 2-µm-diameter circle spot in a nucleus was bleached, and the fluorescence recovery was measured. RNAP2 Ser2ph-mintbody fluorescence recovered to 80% within 2 s in untreated cells, and the recovery speed increased (to <1 s) in cells treated with flavopiridol and triptolide for 2–4 h (n = 35).

Evaluating the specific binding of the RNAP2 Ser2ph-mintbody to RNAP2 Ser2ph in living cells. (A and B) HeLa cells that stably express the RNAP2 Ser2ph-mintbody were treated with 1 µM flavopiridol, 5 µM triptolide, or vehicle (0.1% DMSO) for 2 h. High-resolution confocal images were acquired using a high-resolution confocal system. (A) Representative images at 0, 60, and 120 min. Scale bar, 5 µm. (B) Nuclear foci were selected using autothresholding, and the total area in the single nucleus was measured. The areas relative to time 0 were plotted with SEM (n = 10). The area of foci decreased by treatment with flavopiridol and triptolide. (C) FRAP. A 2-µm-diameter circle spot in a nucleus was bleached, and the fluorescence recovery was measured. RNAP2 Ser2ph-mintbody fluorescence recovered to 80% within 2 s in untreated cells, and the recovery speed increased (to <1 s) in cells treated with flavopiridol and triptolide for 2–4 h (n = 35).

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