Quantifying Protein Mobility and Immobile Fractions

For FRAP analysis analysis^{69 (link),71 (link)}, the GFP-fluorescence signal of our GFP-tagged proteins was measured in a strip across the nucleus (width 512 × 16 pixels, zoom ×12), at 1400 Hz of a 488 nm laser every 22 ms until a steady-state was reached (pre-bleach). Using 100% power of the 488 nm laser, the fluorescent signal in the strip was bleached and fluorescence recovery was monitored every 22 ms until recovery was complete. All FRAP experiments were acquired on a Leica TCS SP5 microscope (with LAS AF software, Leica, version 2.7.4.10100) equipped with a 40x/1.25 NA HCX PL APO CS oil immersion lens (Leica Microsystems), at 37 °C and 5% CO₂. Fluorescence signals were normalized to the average pre-bleach fluorescence after background signal subtraction. For the quantification of the immobile fractions (F_imm), shown in Fig. 1b, d; 5h, i; Supplementary Fig. 1j, 6, the average recovered fluorescence intensity of UV-irradiated cells (I_final,UV) was divided by the average recovered fluorescence intensity of unchallenged cells (I_final,unc) over the last 10 s of the measurements, after correction with the fluorescence intensity recorded immediately after bleaching (I₀)^{69 (link)}: $\mathrm{Fimm}=\mathrm{1}-\frac{\mathrm{Ifinal,\; UV}-\mathrm{I0,\; UV}}{\mathrm{Ifinal,\; unc}-\mathrm{I0,\; UV}}.$