MADD is a GEF for secretory Rabs. (A) MADD expression was analyzed by quantitative polymerase chain reaction 7 days after transduction of HUVECs with shCTRL or shMADD. Data are from 3 biological replicate experiments. (B) Pulldown (PD) of active, GTP-bound Rab27A using GST-Slac2-b-SHD (Slac2-b)-coupled beads in shCTRL- and shMADD-transduced HUVEC lysates. GTP-bound Rab3B (C) and Rab3D (D) were extracted using GST-RIM2-RBD (RIM2)-coupled beads. PD experiments were performed in biological triplicate, and representative Western blots are shown. (E-G) Bar graphs represent normalized active fractions of Rab27A (E), Rab3B (F), and Rab3D (G) relative to total input levels. Data are shown as mean ± standard error of the mean [SEM] and analyzed using unpaired two-tailed Student t test. Dotted lines indicate activity levels in shCTRL. *P < .05; **P < .01.