Ce-Punctin/MADD-4 localizes SDN-1 at synapses. (A) Structure of the madd-4 locus. Boxes represent exons. Arrows indicate the beginning of the ORF of madd-4L and madd-4B. Localization of isoform specific mutations (ttTi103747 and tr185) and full knockout (kr270), referred to as madd-4(0), are indicated. (B) Confocal detection of mNG-SDN-1 and cholinergic active zones (CLA-1-BFP). (C) Confocal detection and fluorescence profiles of mNG-SDN-1 and GABAergic boutons using the SNB-1-BFP marker driven by the Punc-47 promoter. (D) Quantification of mNG-SDN-1 fluorescence levels. (E) Pearson’s correlation coefficient between mNG-SDN-1 and GABA boutons. (F and G) Confocal detection and quantification of MADD-4-RFP fluorescence. (H) Coimmunoprecipitation of SDN-1 and MADD-4B expressed in HEK293 cells. Cells were transfected with HA-SDN-1 alone (lanes 3 and 6), MADD-4B-GFP (lanes 1 and 4), or GFP (lanes 2 and 5). Cell lysates were precipitated by GFP nanobody beads and probed with anti-HA antibody (upper). The expression of GFP was detected using anti-GFP antibody (lower). M, protein ladder. Molecular weight (Mw) is shown on the right. Scale bars, 10 µm.