Figure 2.

Interaction between NCAM and D₂R in cells and tissue. A, Cultured hippocampal neurons were subjected to immunostaining with NCAM antibody and D₂R antibody; nucleus was stained with DAPI. Colocalization of NCAM and D₂R in cell soma and neurites is indicated by the white arrows. Scale bar, 20 μm. B, Brain homogenate, synaptosomal fraction, or HEK293 cell lysate transfected with D₂R and NCAM were subjected to immunoprecipitation (IP) using mouse anti-D₂R antibody or nonspecific mouse IgG. Precipitated proteins were analyzed by Western blot with NCAM antibody 5B8. Coimmunoprecipitation of NCAM180 and D₂R was observed. C, NCAM180-ICD, NCAM140-ICD, or CHL1-ICD were coupled to the biotin containing cross-linker Sulfo-SBED and incubated as baits with brain homogenate under dephosphorylation (P−) or phosphorylation (P+) conditions. After UV cross-linking and denaturation under reducing conditions, the biotin moiety was transferred from the cross-linker to the molecules bound to the baits. Bound proteins were analyzed by Western blot (WB) using Neutravidin and anti-D₂R antibody. D, Myc-D₂R-expressing HEK293 cells were transfected with NCAM180 and stimulated with 10 μm DA for 0, 10, or 60 min. Cell lysates were prepared and subjected to immunoprecipitation (IP) using mouse anti-D₂R antibody. Precipitated proteins were analyzed by Western blot with NCAM antibody 5B8. Coimmunoprecipitation of NCAM180 and D₂R was observed. The amount of coimmunoprecipitated NCAM was quantified and the amount obtained after 0 min stimulation was set to 100%. Means + SEM are shown. *p < 0.05 by unpaired t test (n = 3).