MADD activates secretory Rabs in a DENN domain–dependent manner. HEK293T cells were transfected with GFP-Rab27A (A), GFP-Rab3B (B), or GFP-Rab3D (C) alone or co-transfected with GFP-MADD-full-length or GFP-MADD-ΔDENN. GTP-bound Rabs were extracted from lysates using Slac2-b or RIM2 PD as indicated. GFP-tagged proteins were visualized using an anti-GFP antibody. Experiments were performed in technical duplicates, and representative Western blots are shown. (D-F) Bar graphs represent mean normalized active fractions of Rab27A (D), Rab3B (E), and Rab3D (F) relative to input levels. Dotted lines indicate activity levels in cells transfected with GFP-Rabs without GFP-MADD variants.