(A-D) Neutral sphingomyelinase (nSMase)-RNAi was expressed using the flight muscle driver Act88F-GAL4 in Gba1b mutants and wildtype revertant controls. Isolated EVs from these flies were prepared in RIPA buffer. Quantification of western blot analysis of (A) Ref(2)P (one-way ANOVA: F(3,8) = 20.397, p < 0.001), (B) ubiquitin (F(3,8) = 55.25, p < 0.001), (C) Rab11 (F(3,8) = 6.713, p = 0.014), and (D) Rab7 (F(3,8) = 5.159, p = 0.028) in the EV fraction are shown. Results are normalized to control. At least 3 independent experiments were performed. Error bars represent SEM. *p < 0.05 by Student t-test. (Representative images are found in S1 Fig).