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. 2021 Apr 1;40(10):e105464. doi: 10.15252/embj.2020105464

Figure EV4. Single‐molecule microscopy analysis of Myc mutants.

Figure EV4

  1. Immunoblots on subcellular fractions, used for the quantifications shown in Fig 4A. Cyt, Nuc, and Chr indicate the cytoplasmic, nucleoplasmic, and chromatin‐associated fractions, respectively, all loaded with the same cell equivalents as the total. Histone H3 and Vinculin were used as markers of the Chr and Cyt fractions, respectively. Note that the spread pattern of H3 in the central blot is most likely caused by its presence close to the front of migration in SDS–PAGE. In the first gel, the loading of the last two lanes was inverted, as indicated by the names in red. Saturated: over‐exposure of the blot, shown in order to visualize weak bands.
  2. Schematic representation of the illumination protocol for the SMT acquisitions to quantify the fraction of bound Myc molecules and exemplary acquisitions. The maximal projection of a representative MycWT movie is shown, displaying the nucleus boundaries (cyan dotted line) and a representative region (yellow square), for which exemplary frames are displayed on the right. The blue and red arrows highlight a bound and a diffusing molecule, respectively. Scale‐bar: 5 µm.
  3. Tracking the single Myc molecules allows estimating the distribution of displacements which is fit with a three‐component diffusion model to extract the fraction of bound molecules as well as the fractions and the diffusion coefficients of the diffusing molecules (see Materials and Methods).
  4. Schematic of the illumination protocol for the SMT movies to quantify the residence times of bound Myc molecules. Acquisition at different frame rates (ttl ranging between 200 ms and 2s) is performed to measure the residence times of bound Myc molecules at multiple time‐scales and to correct for photobleaching.
  5. The cumulative distributions of residence times are analyzed together using a global model accounting for photobleaching.
  6. The model allows estimating three characteristic times τ 1,τ 2,τ 3— inverse of the three characteristic reaction rates, whose weighted average provides an estimate of the mean residence time of the various Myc proteins on chromatin (See Materials and Methods and Fig 4C).