Figure 8.

SynCAM 1 and 2 form a specific adhesive complex in synaptic membranes in vivo. Synaptosomes were prepared from rat forebrain at P15 during the peak of SynCAM expression and synaptogenesis. Synaptosomal membrane proteins were extracted with detergent (lane 1) and immunoprecipitated with specific antibodies against the extracellular domain of SynCAM 1 (lanes 2, 3) or control antibodies (lanes 4, 5). The flow-through (FT) of each immunoprecipitation was obtained, and immunoprecipitates (IP) were collected on beads, washed, and eluted with SDS. The same amount of antibody used for the immunoprecipitation was loaded to identify cross-reactive bands recognized by secondary antibodies (lane 6), which are marked by asterisks. Fractions were probed for each of the four SynCAM proteins as indicated, and with an antibody detecting the L1 Ig superfamily member and the synaptic vesicle protein synaptophysin as negative control membrane proteins. SynCAM 1 coimmunoprecipitated SynCAM 2 from synaptosomes but no other SynCAM family member (lane 3). Numbers on the left indicate positions of molecular weight markers. Input and FT lanes contain 4% of the extract amount used for each immunoprecipitation.