Fig 1: WASH-mediated protein trafficking prevents lysosomal degradation of SG proteins.(A) Transcript levels of Pcsk1, Pcsk2, Chgb, and Cpe in 8-week-old Magel2p?/m+ and Magel2+/+ mouse hypothalamuses, n > 3 per genotype. Each data point represents 1 animal, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test, ****P < 0.0001. (B) Transcript levels of PCSK1, PCSK2, CHGB, and CPE between control and PWS iN at 14 days postinduction. Each data point represents 1 induction experiment (n = 3), plotted as mean ± SD and analyzed by 1-way ANOVA. (C) Western blot analysis shows rescued expression of PCSK1, PCSK2, CHGB, and CPE in PWS iN at 14 days postinduction following inhibition of lysosomes by Baf (1 µM, 7 hours) but not inhibition of proteasomes by MG132 (10 µM, 7 hours). DMSO served as vehicle control. GAPDH served as loading control. (D) Quantification of Western blot analysis confirmed reduced expression of Pcsk1 and Pcsk2 in 2-week-old Washc1 conditional knockout (cKO) mouse hypothalamuses, n = 4 per genotype. Gapdh served as loading control. Each data point represents 1 animal, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test, *P < 0.05. (E) Representative images of immunofluorescence staining with CHGB and WASH at both soma and neurite in control iN. Scale bars indicate 10 µm.