Quantifying Protein Mobility Using FRAP

For FRAP, cells were seeded on coverslips and imaged with a Leica TCS SP5 or SP8 microscope using a 40x/1.25 NA HCX PL APO CS oil immersion lens (Leica Microsystems) at 37 °C and 5% CO₂. FRAP was performed as described^{46 (link),109 (link),110 (link)}. Briefly, the nuclear fluorescent signal of the GFP-tagged protein was measured in a strip across the nucleus (512×16 pixels) at 1400 Hz of a 488 nm laser until a steady state level was reached (pre-bleach). The fluorescent signal in the strip was bleached with 100% 488 nm laser power and fluorescence recovery was measured until complete recovery. Fluorescence signals were normalized to the average pre-bleach fluorescence after background signal subtraction. The immobile fractions (F_imm) were determined using the fluorescence intensity measured immediately after UV (I₀) and the average steady-state fluorescent signal after complete recovery, from untreated (I_{final, unt}) and UV-treated cells (I_{final, UV}), and applying the formula: $\mathrm{F}\mathrm{imm}=1-\frac{\mathrm{I}\mathrm{final},\mathrm{UV}-\mathrm{I}0,\mathrm{UV}}{\mathrm{I}\mathrm{final},\mathrm{unt}-\mathrm{I}0,\mathrm{UV}}$