(A) Synaptosomes from mouse hippocampi from P9, P21, and adult (P55) were immunoblotted for indicated proteins. lys; lysate. syn; synaptosome. 2 μg protein per lane. (B, C) 14 days in vitro (DIV) cultured hippocampal neurons immunostained with antibodies against Kirrel3 (red), vGlut1 (green), and MAGUK proteins (blue). Boxed regions in B are magnified below in B′, B′′, and B′′′. Neurons from Kirrel3 knockout mice have no Kirrel3 signal (C). (D) Diagram of Kirrel3 protein and location of inserted FLAG tag. Ig; immunoglobulin. (E–H) Cultured hippocampal neurons were co-transfected with FLAG-Kirrel3 and GFP and immunostained for indicated proteins. Anti-FLAG antibodies were added prior to fixation to label only surface Kirrel3. Note surface Kirrel3 is seen as puncta on axons and dendrites after synapse formation in 14DIV neurons (E–G) and prior to synapse formation in 4DIV neurons (H). H shows that surface Kirrel3 also clusters at axon–dendrite crossings. Boxed regions in E are shown magnified in F and G. FLAG signal alone is shown in lower panels F′, G′, and H′. (I, J) Kirrel3 clusters at cell junctions. 293HEK cells were co-transfected with GFP and FLAG-Kirrel3, immunostained for GFP and FLAG, and nuclei labeled with Hoechst. (K–M) CHO cells transfected with either GFP control or GFP and Kirrel3 were tested for adhesion. Only cells expressing Kirrel3 formed aggregates. Aggregation index was calculated by dividing the total GFP fluorescence in cell aggregates by the total GFP fluorescence in the well. Mean ± SEM are shown, n = 3, *** indicates p = 0.001 by two-tailed t-test.