Effects of thresholding parameters on the difference between median D values in molecules highly and lowly associated with RNAP2 Ser2ph-mintbody-enriched regions. (A and B) The difference between median D values in HaloTag-tagged RPB3 molecules that were highly and lowly associated with RNAP2 Ser2ph-mintbody–enriched regions were analyzed by altering the thresholding parameters to define the fractions. The percentages of molecules to yield the top and bottom fractions of RNAP2 Ser2ph-mintbody intensity were changed from 5% to 45%; note that 25% was used for the detailed analysis. The D_thr to define bound fraction was also changed from 0.025 to 0.169 µm²/s; note that 0.065 µm²/s was used for the detailed analysis. σ_D = 2.0 indicates D_thr with the mean and −2 SD of the mobile fraction. After applying the thresholds, the difference between median D values of fractions with the top and bottom RNAP2 Ser2ph-mintbody intensity (D_top and D_bottom, respectively; A) and the number of molecules (B) are plotted with the P values derived by Mann-Whitney U test and the numbers of analyzed bound molecules (n). In a broad range of D_thr (0.045–0.169 µm²/s), significant differences between median D_top and D_bottom were observed in any percentages from 5% to 45%, with the lowest P values in the range of 20–30%.