![Generation of the RNAP2 Ser2ph-specific mintbody and characterization of the mintbody foci in HeLa cells.(A) Schematic diagram of the mintbody that binds to phosphorylated Ser2 on the CTD of RNAP2. (B) Single confocal sections of 42B3(original)-mCherry and 42B3(R78K/A80T/M95V)-mCherry, which were stably expressed in HeLa cells. Fluorescence images were acquired using a confocal microscope under the same settings, and image contrast was enhanced differently [70–500 for the original 42B3-mCherry and 70–2,000 for 42B3(R78K/A80T/M95V)-mCherry] because the intensity of the former was dimmer. (C) N/C ratios of 42B3 and mutants tagged with mCherry. HeLa cells expressing 42B3-mCherry and mutants were established. After confocal images were acquired, the N/C ratios of fluorescence were measured (n = 30). In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; data points are plotted as open circles. (D–F) RNAP2 Ser2ph-mintbody in living HeLa cells. (D) Using a high-resolution spinning-disk confocal system, the fluorescence image of RNAP2 Ser2ph-mintbody in a living HeLa cell was acquired. A single section of the confocal image is shown. Bottom right: An example of focus with a 1-µm line in the indicated area. (E) Estimating the size of the foci. Line intensity profiles of single fluorescence foci were plotted in a.u. Top: An example of the focus and its line intensity profile with the fitted curve. Top right: The intensity plot of the line and the fitted curve by the Gaussian distribution model. Bottom: Average fitted curves from 20 spots per cell are shown with an average full width at half maximum (FWHM) for three different cells. (F) Estimating the number of foci. Foci that were identified by a spot detection algorithm are indicated by green circles. A magnified view is shown in the middle. The total number of foci was counted by analyzing the z-sections covering the whole nucleus at 0.2-µm intervals. As a single focus can be detected in a maximum of three sections, the total counts were divided by 3. The numbers and average intensities (a.u.) of foci in 68 cells are plotted. Scale bars, 5 µm; 1 µm for magnified views.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/2/10.1083_jcb.202104134/4/jcb_202104134_fig1.png?Expires=1771219374&Signature=z6R~WwrF9BystXQYunYvHQx2FlNuSIcC2eV7vEFgFPP7X-1W25JvXb6GAJP7pqcLGOc-09HhW~lNoKuXerwdhM1WWE7IvRPru9vGrHT47dFg4h3viEWgdHHJkBNnzV67J7iyDgvCFN2nryL8HTAYmoXKRlO6kJ1D1pUV0jDl4bZ8H38Wdm71p7houPYPUqIcJXzGW~XqNkhOfaRBfE8cysJnz8EXJH00yd9Pmx4MMWSIw-B3O7o1TOhCd~OK1CxLTzxdIGMTX7Nddb-3mYLa6o0skh6oF60JeMC-Yrb2uqA45LvAzOYae5oMHlxlLp~mZX8H~2Ykh4YfoSGa3EQAyw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Generation of the RNAP2 Ser2ph-specific mintbody and characterization of the mintbody foci in HeLa cells. (A) Schematic diagram of the mintbody that binds to phosphorylated Ser2 on the CTD of RNAP2. (B) Single confocal sections of 42B3(original)-mCherry and 42B3(R78K/A80T/M95V)-mCherry, which were stably expressed in HeLa cells. Fluorescence images were acquired using a confocal microscope under the same settings, and image contrast was enhanced differently [70–500 for the original 42B3-mCherry and 70–2,000 for 42B3(R78K/A80T/M95V)-mCherry] because the intensity of the former was dimmer. (C) N/C ratios of 42B3 and mutants tagged with mCherry. HeLa cells expressing 42B3-mCherry and mutants were established. After confocal images were acquired, the N/C ratios of fluorescence were measured (n = 30). In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; data points are plotted as open circles. (D–F) RNAP2 Ser2ph-mintbody in living HeLa cells. (D) Using a high-resolution spinning-disk confocal system, the fluorescence image of RNAP2 Ser2ph-mintbody in a living HeLa cell was acquired. A single section of the confocal image is shown. Bottom right: An example of focus with a 1-µm line in the indicated area. (E) Estimating the size of the foci. Line intensity profiles of single fluorescence foci were plotted in a.u. Top: An example of the focus and its line intensity profile with the fitted curve. Top right: The intensity plot of the line and the fitted curve by the Gaussian distribution model. Bottom: Average fitted curves from 20 spots per cell are shown with an average full width at half maximum (FWHM) for three different cells. (F) Estimating the number of foci. Foci that were identified by a spot detection algorithm are indicated by green circles. A magnified view is shown in the middle. The total number of foci was counted by analyzing the z-sections covering the whole nucleus at 0.2-µm intervals. As a single focus can be detected in a maximum of three sections, the total counts were divided by 3. The numbers and average intensities (a.u.) of foci in 68 cells are plotted. Scale bars, 5 µm; 1 µm for magnified views.
