E3 ligase Ctrip preferentially interacts with glutathionylated Gbb. (A–C) Confocal images of NMJs double-labeled with anti-HRP (green) and anti-CSP (magenta). The genotypes are: WT, ctrip RNAi1 (THU0789)/C57-Gal4, ctrip RNAi2 (TH01600.N)/C57-Gal4. Scale bar, 10 µm. (D) Quantification of total bouton numbers of different genotypes. n = 10 NMJs for each genotype, statistical significance was calculated using one-way ANOVA; ***P < 0.001; error bars denote SEM. (E–G) Confocal images of NMJs colabeled with anti-Gbb (green) and anti-HRP (magenta). Knockdown of ctrip by C57-Gal4 resulted in increased intracellular Gbb level compared with WT. (H) Quantification of cytoplasmic Gbb level of different genotypes in arbitrary units (a.u.). n = 12 larvae; statistical significance was calculated using one-way ANOVA; ***P < 0.001; error bars denote SEM. (I) Gbb preferentially interacts with Ctrip2. Lysates of S2 cells co-expressing Myc-Gbb and individual Flag-tagged Ctrip fragments were subjected to immunoprecipitation with anti-Flag antibody. Gbb was detected with anti-Myc antibody. Tubulin was used as a loading control. (J) Ctrip2 binds weakly to non-glutathionylated C354A and C420A double mutation Gbb. S2 cell lysates co-overexpressing Flag-Ctrip2 and WT or C354A and C420A double mutated Gbb. Myc-Gbb was subjected to co-immunoprecipitation with anti-Flag antibody and detected with anti-Myc antibody. Tubulin was used as a loading control. (K) A working model. GstO1 promotes glutathionylation and subsequent proteasome-mediated degradation of Gbb, while Grx1 does the opposite. GstO1 and Ctrip negatively regulate NMJ synapse development by down-regulating Gbb protein level. Source data are available for this figure: SourceData F10.