Supplemental data

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• supplemental material - Supplementary Figure S1 Comparison of MAG-Fc and AP-MAG binding to NgR2 and NgR1. �A:� Semi-quantitative analysis of MAG-Fc binding to NgR1 and NgR2 transiently expressed in COS-7 cells.� Serial dilutions (0-80 nM) of MAG-Fc (pre-clustered with an anti-human Fc-AP conjugated antibody) were washed on transiently transfected COS-7 cells.� To compare the relative binding strength to NgR1 and NgR2, the AP reaction was developed and followed over time.� After ~1h, labeling of NgR2 transfected cells incubated with MAG-Fc at 5 nM started to become detectable and the AP reaction was stopped in all wells.� In three independent experiments (of a total of three experiments) MAG-Fc bound NgR2 more strongly than NgR1.� MAG-Fc binding to NgR2 at a concentration of 5-10 nM is comparable to MAG-Fc binding to NgR1 at a concentration of 40-80 nM.� Thus, MAG-Fc binds approximately 4-8 fold stronger to NgR2 than to NgR1.� B:� Binding of AP-MAG (17 and 33 nM) to transiently transfected COS-7 cells.� Consistent with a previous study (Barton et al., 2003), AP-MAG binds to NgR1 but not to NgR2.� Thus, N-terminal tagging of MAG with AP selectively abolishes binding to NgR2.� Consistent with this finding, we show that the structural basis of NgR1 and NgR2 to support MAG binding is distinct (see figure 8 for details).�� Scale bar is equal to 10 mm (A and B).
• supplemental material - Supplementary Figure S2 Soluble NgR1 does not interact with NgR2.� Affinity precipitation experiments, using NgR1-Fc (NgR-Fc, R&D Systems) or anti-human IgG did not reveal any interaction between NgR1 and NgR2.� Western blot analysis of lysates of Ad-NgR2 transduced cells (Inp.) revealed robust expression of NgR2.� As a control, we show that NgR1-Fc allows selective precipitation of AP-Nogo66 but not AP-NiG.�