Fig. 3. Zebrafish MAGI1 binds directly and specifically to zebrafish DeltaC and DeltaD. (A) Beads were coated with peptides corresponding to the C-terminal 27 or 26 amino acids of DeltaC (top panel) or DeltaD (bottom panel) either with the terminal valine (V) or without (E). The beads were then incubated with purified proteins corresponding to each of the six individual PDZ domains of zebrafish MAGI1 (0 to 5). Beads were then washed and bound proteins eluted and resolved by SDS-PAGE under reducing conditions. Both DeltaC and DeltaD peptides that terminated in the ATEV motif interacted directly and selectively with the PDZ4 domain alone, while control peptides that lacked the terminal valine (ending –ATE) showed no binding to any PDZ domain. (B,C) HEK293T cells cotransfected with plasmids coding for MAGI1-EGFP (green) and either full-length DeltaD (red in B) or DeltaD lacking its terminal valine (DeltaD-TE^*; red in C). Both forms of DeltaD were detected with zdd2 anti-DeltaD monoclonal antibody. DeltaD, but not DeltaD-TE^*, recruits MAGI1 to the plasma membrane [compare regions indicated by arrows in (B) and (C)]. 56 out of 59 cells transfected with DeltaD showed MAGI1 membrane recruitment, but only 1 out of 37 transfected with DeltaD-TE^* did so. (D,E) Confocal sections of somite cells in 10-14 somite stage zebrafish embryos injected with 20 pg mRNA encoding for MAGI1-EGFP at the two- to four-cell stage, either (D) alone or (E) with coinjection of 5 ng MO[dlD-V]. MAGI1-EGFP was detected with an anti-GFP antibody and endogenous DeltaD was detected with zdd2. Note co-localization of DeltaD and MAGI1 in (D) but not (E). Scale bars: 10 µm.