Fonseca et al., Supplementary Information FRAP data analysis

Supplementary Legends
Fonseca, Steffen, Müller, Lu, Sawicka, Seiser and Ringrose
Figure S1. Diffusion influences FRAP recovery for GFPnls, PC::GFP, PH::GFP and
H2A::RFP. Diffusion test was performed using an adaptation of the method of curve
smoothing (Mueller et al. 2008). (A-F) Radial intensity profiles of FRAP experiments at
four different time points after photobleaching (time in seconds is shown at the right of
each plot). The gaussian edges of intensity profiles normalized to prebleach levels
(Mueller et al. 2008) are plotted (symbols) and were fitted using linear regression (solid
lines). The gray background indicates the bleach region. (A) H2A::RFP recovery is not
affected by diffusion, indicated by similar slopes of lines at all four time points.
Comparison of the extracted slopes was performed using ANCOVA (p-value given on
each plot represents significance of difference between slopes at the four time points).
(B-F) GFP-nls, PC::GFP and PH::GFP FRAP recovery shows an influence of diffusion,
indicated by gradual flattening of radial profiles at later time points. (E) Comparative
summary plot. For data in (A-F), the value 1/slope was calculated for each linear fit
and normalized to the slope at time 0. These values are plotted for each data set for
consecutive time points, showing a gradual increase in (1/slope) at later time points for
all experiments with the exception of H2A::RFP (black) for which little change was
detected.
Figure S2. Comparison of the effects of binding site non-homogeneity on parameters
extracted from FRAP experiments. Extracted diffusion (A,D,G and J), free fraction
(B,E,H and K) and dissociation rates (koff, C,F,I and L) of PH::GFP (A-C, G-I) and
PC::GFP (D-F, J-L) FRAP experiments in neuroblast interphase (A-F) and sensory
organ precursor cell interphase (G-L) were analysed using an adaptation of the model
described in (Mueller et al. 2008). (See Supplementary Information, FRAP Data
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