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Fig. 5. Aggregation, but defective postsynaptic clustering, of EGFP-geph-GCEE in HEK293 cells and neurons. (A) Aggregates of EGFP-geph-GCEE (green) in HEK293 cells without colocalization with 
1 subunit immunoreactivity (red) on the cell surface. (B) EGFP-geph-GCEE formed aggregates in 6+2 cells, located in the soma and in dendrites and lacking 2 subunit staining (arrowheads). (C) In 6+6 cells, only few EGFP-positive clusters colocalized with 2 subunit immunoreactivity were detected (open arrowheads). Cytosolic aggregates (arrowheads) were larger and more numerous. (D) In contrast to EGFP-geph-GCE, EGFP-geph-GCEE was not integrated in all gephyrin-immunoreactive clusters (open arrows); (E) Only a few EGFP-geph-GCEE-positive clusters associated with the 2 subunit and apposed to a synapsin-1-positive presynaptic terminal (blue) were observed (open arrowheads in E''); aggregates (green) were devoid of 2 subunit staining and were not postsynaptic (arrowheads in E''). (F-H) Three examples of cortical neurons transfected by nucleofection with EGFP-geph-GCEE, showing different aggregation and clustering in dependence of expression levels. In cells containing numerous aggregates (F; arrows), only few postsynaptic clusters were present (open arrowhead in f1); clusters of endogenous gephyrin were present in reduced numbers (arrowhead). When aggregates were few and small (G,g1; arrows), a larger number of postsynaptic clusters (g1, open arrowheads) was seen; finally, almost normal postsynaptic clustering was evident (H,h1; open arrowheads) when aggregates were nearly absent (arrow). Bars, 10 µm (A-C,F-H); 5 µm (D,E).
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