Figure 3. Disruption of OPHN1 Interactions with Homer 1b/c and Endo2/3.

(A) Domain structure of OPHN1. The Homer 1b/c and Endo2/3 binding sites and the R409Q amino acid substitution in GAP domain that abolishes OPHN1’s Rho-GAP activity are indicated.

(B) Homer 1b-GST fusion protein, or GST alone, immobilized on beads was incubated with extracts from HEK293T cells expressing OPHN1^WT-EGFP or OPHN1^Hom-EGFP. Bound OPHN1 was detected by immunoblotting with anti-GFP antibody. GST-fusion proteins used are indicated by Coomassie Blue (CBB) staining. TL, total lysate.

(C) Homer 1b-GST fusion protein (lower panel, CBB staining) immobilized on beads was first incubated with synaptosomal extracts followed by pep-OPHN1^Hom or pep-cont^Hom at indicated concentrations. Bound OPHN1 was detected by immunoblotting with anti-OPHN1 antibody.

(D) Endo2-GST fusion protein, or GST alone (lower panel, CBB staining), immobilized on beads was incubated with extracts from HEK293T cells expressing OPHN1^WT, OPHN1-PRD1*, -PRD2*, or -PRD3*(= OPHN1^Endo)-EGFP fusion proteins. Bound OPHN1 was detected by immunoblotting with anti-GFP antibody.

(E) Extracts prepared from acute hippocampal brain slices (CA1 regions), pretreated with cycloheximide (CHX, 50 µM), or control vehicle, for 5 min and then treated with DHPG (100 µM), or ACSF, for 10 min were incubated with anti-Endo2 antibody, or control IgG. The immunoprecipitates (IP) and total lysates (TL) were analyzed by immunoblotting with indicated antibodies. Relative amount of co-immunoprecipitated OPHN1 with Endo2 (compared to control vehicle) is shown in right panel. n = 3, *p < 0.01 (unpaired t-test), as compared with control vehicle-treated slices. Error bars represent SEM.

(F) Endo2-GST fusion protein (lower panel, CBB staining) immobilized on beads was first incubated with synaptosomal extracts followed by pep-OPHN1^Endo or pep-cont^Endo at indicated concentrations. Bound OPHN1 was detected by immunoblotting with anti-OPHN1 antibody.