Fig. 3

Identification of NCAM1 as a novel neuronal MHC-I interacting protein. A Proteins in synaptosomes from normal aged human brain that interacted with MHC-I, as shown by coimmunoprecipitation with W6/32 antibody and staining with Coomassie blue. Gel slices 1–7 were processed by in-gel digestion and LC–MS/MS analysis. B Confirmation of the MHC-I–NCAM1 interaction by immunoblotting with antibodies to MHC-I and NCAM1. Synaptosome purity was assessed by immunoblotting with antibodies to PSD95, synaptophysin, and GAPDH. C, D Coimmunoprecipitation analysis of the interaction between NCAM1 and MHC-I. Lysates of SH-SY5Y cells expressing NCAM1-hemagglutinin (HA) were immunoprecipitated with antibodies to HA (C) and W6/32 (D), followed by immunoblotting with the anti-HA and anti-MHC-I antibodies (C, D). The blots are representative of three independent experiments. E Bio-Layer Interferometry analysis of the affinity of interaction between MHC-I and NCAM1. NCAM1 protein (100 nM) was immobilized onto a biosensor, which was incubated with recombinant biotinylated MHC-I–β₂M complex (HLA-A2.1 allele) at concentrations of 0, 50, 100, 250, 500, and 1000 nM. All data are presented as the mean ± SEM (*P < 0.05, **P < 0.001, ***P < 0.005 by unpaired two-tailed Student’s t-test)