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. 2023 Oct 27;5(6):fcad295. doi: 10.1093/braincomms/fcad295

Figure 2.

Figure 2

Functional studies of the ADAM22 protein with S905F variant. (A and B) The interaction of ADAM22 S905F with PSD-95 is significantly reduced. Indicated cDNAs for ADAM22 variants and PSD-95–FLAG were co-transfected into HEK293T cells and PSD-95–FLAG was immunoprecipitated (IP). ADAM22 and PSD-95 were detected by western blotting (WB). ADAM22ΔC5, lacking the C-terminal PDZ-binding motif, is used as a control.8 Arrows and arrowheads indicate the positions of immature and mature forms of ADAM22, respectively. The immature form of ADAM22 is often observed in overexpressed cells. P values were determined by Kruskal–Wallis with post hoc Steel test. *P < 0.05; n.s., not significant; n = 3 experiments. Results are shown as means ± SE. WT, wild-type. See Supplementary material for uncropped blots for A. (C) The S905F variant reduces the binding of ADAM22 to MAGUK proteins including PSD-95, PSD-93 and SAP102 in HEK293T cells. Conserved Asn and Lys residues are shown in blue. (D and E) ADAM22 S905F binds to LGI1. ADAM22 C401Y is used as a control, which shows the reduced binding to LGI1.7,14  P values are determined by Kruskal–Wallis with post hoc Steel test. *P < 0.05; n = 3 experiments. Results are shown as means ± SE. Immature ADAM22 (arrows) seems to non-specifically bind to LGI1 under the overexpressed conditions. In the brain, we do not detect such an immature form of ADAM22. See Supplementary material for uncropped blots for C and D. (F and G) ADAM22 S905F is expressed at the cell surface and binds to LGI1. Cell-surface-expressed ADAM22 (F, magenta) and cell-surface-bound LGI1 (G, magenta) were live-labelled. After fixation and permeabilization of the cells, expressed ADAM22 (total) was stained (green). Nuclear DNA was stained by Hoechst 33342 (F and upper panel in G, blue). ADAM22 S905F is expressed on the cell surface and binds to LGI1 as the wild-type. When ADAM22 C401Y is expressed, no cell-surface-bound LGI1 is detected. Regions outlined with white squares are total expression of ADAM22 (F) and LGI1–FLAG (lower panel in G, blue). Bars: 20 μm.