Fig. 3.

The RECI model correctly recapitulates a FRAP experiment. (A) After 1 h of TGF-β treatment, nuclear EGFP-Smad2 was photobleached and nuclear fluorescence recovery was monitored. Representative pictures of a recovery time course are shown. (B) To model the FRAP recovery, simulations were run for both models until maximal response to TGF-β was reached. The simulation was stopped, and from these initial conditions (RECI model: 68 nM EGFP-Smad2 in the nucleus, 42 nM in the cytoplasm; RO model: 64 nM in the nucleus, 45 nM in the cytoplasm), a photobleach was simulated by converting all nuclear species containing EGFP-Smad2 into the corresponding species containing endogenous, i.e., “invisible” Smad2. The simulation was continued, and the sum of the concentrations of all nuclear fluorescent species was plotted. Because the experimental data were measured in arbitrary units, a least-square method was used to scale the experimental dataset (○; ± SD, n = 10) such that maximal overlap with either the RECI model (red line and red ordinate) or the RO model (black line, black ordinate) was achieved. Note the superior performance of the RECI model when compared with the RO model in capturing the shape of the recovery curve. (C) The recovery curve (red line) is a sum of the recovery behavior of all species containing EGFP-Smad2, whose concentrations were calculated by using the RECI model.