Cell, Volume 130
A Vast Repertoire of Dscam Binding
Specificities Arises from Modular
Interactions of Variable Ig Domains
Woj M. Wojtowicz, Wei Wu, Ingemar Andre, Bin Qian, David Baker,
and S. Lawrence Zipursky
Figure S1. Comparison of Binding Assays to Assess Dscam Binding Specificity
The binding properties of Dscam isoforms containing Ig2.7, Ig3.27 and highly-related Ig7 domains are
shown in four different binding assays. The table outlines the properties of each binding assay to indicate
1) sensitivity (as determined by whether heterophilic binding can be detected between isoforms 7.27.25
and 7.27.26), 2) whether the assay is readily quantitative, 3) whether the assay requires protein
purification, and 4) the number of binding experiments that can be conducted per day in our hands.
Representative binding data for these isoforms in each assay are shown (Matthews et al., 2007;
Wojtowicz et al., 2004)(J.J. Flanagan and S.L.Z, unpublished observations). F.L., full length.
Figure S2. Detection of Homophilic Binding Requires Clustering of Dscam Molecules.
(A) Immunoprecipitation assay to assess Dscam homophilic binding. Purified Dscam-Fc (bound to
protein G sepharose) was used as receptor protein to pull-down purified Dscam-His ligand protein. Pull-
downs were performed 1) in the absence (lanes 2-5) and presence (lanes 6-9) of Dscam-Fc receptor and 2)
in the absence (lanes 2-3, 6-7) and presence (lanes 4-5, 8-9) of α-His IgG-HRP which was used for
clustering Dscam-His ligand protein (Note that α-His IgG did not bind to protein G sepharose). (B)
ELISA-based binding assay to assess Dscam homophilic binding. Mouse α-Fc IgG was adsorbed to
ELISA plates and used to capture Dscam-Fc receptor protein from cell culture medium following
transient transfection. Dscam-AP ligand containing culture medium was added with or without α-AP IgG
for clustering Dscam-AP. Binding of Dscam-AP ligand to Dscam-Fc receptor was assessed by monitoring
AP activity following addition of AP substrate. This is a variation on the ELISA-based assay used
throughout this paper. AP activity was only linear over a 25-fold range while HRP activity was linear
over a 70-fold range. Additionally, more sensitive reagents are commercially available for HRP detection.
Therefore, the ELISA-based binding assay was optimized such that Dscam-AP is used as receptor and
Dscam-Fc is used as ligand with α-Fc IgG-HRP for clustering and detection of binding (see Figure 1 and
Andre, I., Bradley, P., Wang, C., and Baker, D. (2007). Prediction of the Structure of Symmetrical Protein
Assemblies. PNAS in press.
Das, R., Qian, B., Raman, S., Vernon, R., Thompson, J., Bradley, P., Khare, S., Tyka, M. D., Bhat, D.,
Chivian, D. C., et al. (2007). Structure prediction for CASP7 targets using extensive all-atom refinement
with Rosetta@home. Proteins: Structure, Function and Bioinformatics in press.
Matthews, B. J., Kim, M. E., Flanagan, J. J., Hattori, D., Clemens, J. C., Zipursky, S. L., and Grueber, W.
B. (2007). Dendrite sef-avoidance is controlled by Dscam. Cell 129, 593-604.
Meijers, R., Puettmann-Holgado, R., Skiniotis, G., Liu, J.-H., Walz, T., Wang, J.-H., and Schmucker, D.
(2007). Structural Basis of Dscam Isoform Specificity. Nature submitted.
Wojtowicz, W. M., Flanagan, J. J., Millard, S. S., Zipursky, S. L., and Clemens, J. C. (2004). Alternative
splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic
binding. Cell 118, 619-633.