Figure 4

Neurocan binds the Ig2 domain of NCAM, decreasing EphA3 binding. (A) Fc-pulldowns of the NCAM extracellular domain (NCAM-EC), truncation mutants of NCAM, or control Fc and recombinant neurocan. (B) Densitometry of (A) indicating the level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) for each construct (*p < 0.05 compared to NCAM-EC-Fc) (C) Fc-pulldowns of NCAM-EC-Fc or control Fc with mouse neurocan (lacking sushi domain) and full-length human neurocan. (D) Co-immunoprecipitation of WT NCAM-140 or mutants of NCAM and neurocan from transfected HEK293T cells. (E) Densitometry of (D). The amount of co-immunoprecipitated neurocan for each NCAM IP was normalized to control WT NCAM-bound neurocan (n = 3, t-test, *p < 0.05). (F) Immunoblot of untreated or chABC-treated neurocan protein probed for neurocan or C-4-S. (G) Co-immunoprecipitation of NCAM-140 and EphA3 from transfected HEK293 cells treated with no neurocan (control), neurocan, or chABC-treated neurocan. (H) Densitometry of (G). The amount of co-immunoprecipitated EphA3 for each NCAM IP was normalized to control NCAM-bound EphA3 (n = 3, t-test, *p < 0.05).