Diffusion coefficient and effective spring coefficient of HaloTag-fusion proteins and RNAP2 Ser2ph-mintbody intensity. (A) Scatter plots of the D value and the relative RNAP2 Ser2ph-mintbody intensity (int.). The diffusion coefficients (D) of proteins were obtained using single-molecule trajectories of HaloTag-tagged proteins (RPB3, BRD4, CDK9, p300, H2B, and PCNA) stained with HaloTag TMR Ligand recorded at 33.33 ms/frame, which were superimposed upon high-pass-filtered RNAP2 Ser2ph-mintbody images in living Hela cells (Fig. 7). Scatter plots of D and the relative RNAP2 Ser2ph-mintbody intensity (I_{rel_Ser2ph}) from two independent experiments are shown. Each dot represents a single trajectory (molecule). A vertical line indicates D_thr. The ratio of the bound (D ≤ D_thr) and mobile (D_thr < D) fractions is shown in gray above the plots. The top and bottom 25% I_{rel_Ser2ph} of bound fractions are shown in magenta and green dots, respectively. The median D values of bound molecules of the top and bottom 25% I_{rel_Ser2ph} are also shown. Numbers of trajectories (n) and the rates of bound and mobile fractions are indicated above dot plots. (B) The effective spring coefficient of bound HaloTag-tagged proteins, compared between the molecules that were highly associated (magenta, top 25% I_{rel_Ser2ph}) and lowly associated (green, bottom 25% I_{rel_Ser2ph}) with RNAP2 Ser2ph-enriched regions, with P values derived by Mann-Whitney U test. Box plots from two independent replicate experiments are shown. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by gray dots.