Cell, Volume 130 Supplemental Data Stability and Nuclear Dynamics of the Bicoid Morphogen Gradient

Here we provide further details concerning the experimental methods and implications of the data in Figure 2E-H: E GFP-antibody intensities vs. Bcd-antibody intensities for 15 doubly stained bcd null-mutant embryos expressing Bcd-GFP. Observed linear relationship argues that both the GFP and Bcd regions of the fusion protein are detectable at the same time after translation (both antibodies are attached to different epitopes of the exact same protein) and that the GFP region remains associated with the Bcd region of the fusion protein. GFP does not persist after the Bcd epitope is degraded and miscellaneously cleaved Bcd or GFP protein are present in the egg, suggesting that Bcd and GFP are degraded simultaneously. F GFP-antibody intensities vs. eGFP-autofluorescence intensities for 17 GFP-stained bcd null-mutant embryos expressing Bcd-GFP. The linear relationship is a strong indication that the fluorescence intensities obtained in antibody stainingsfor GFP linearly reflect the actual concentration of eGFP present at a given location in the embryo, and hence via E of Bcd protein. The linearity also suggests that an eGFP maturation delay does not influence Bcd-GFP expression in embryos fixed at this stage of development. The larger scatter around the diagonal compared to E presumably stems from the fact that under these conditions natural eGFP fluorescence is very weak. G Bcd-antibody intensities for 20 randomly selected pairs of embryos from a Bcd-stained batch of 21 wild-type embryos. Linearity implies that this method of comparing profiles is also viable for comparing profiles of embryos of different genotypes with each other. H Bcd-antibody intensities of 14 pairs of embryos randomly chosen from a batch of 15 Bcd-stained embryos expressing Bcd-GFP vs. embryos randomly chosen from a batch of 18 Bcd-stained bcd null-mutant embryos expressing Bcd-GFP. Clustering around the diagonal indicates that the spatial profiles of Bcd-GFP have diffusion and degradation behavior similar to those of the endogenous protein