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. 2018 Sep 13;9:3737. doi: 10.1038/s41467-018-06114-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — MON2-DOPEY2 complex is required for SNX3-retromer-mediated sorting of Wls. a RNAi-mediated suppression of VPS35, SNX3, SNX1 and SNX2, or SNX27 in RPE-1 cells stably expressing HA-WLS and quantification of HA-WLS levels. b Inhibition of lysosomal degradation through Bafilomycin A1 treatment (100 nM for 16 h) restored total protein levels of HA-WLS following RNAi-mediated suppression of VPS35 or SNX3. c RNAi-mediated suppression of VPS35, DOPEY1, DOPEY2, DOPEY1 + DOPEY2 or MON2 reveals that the total protein levels of HA-WLS is dependent on the function of VPS35 and MON2. d Bafilomycin A1 treatment (100 nM for 16 h), inhibiting lysosomal degradation, prevents loss of HA-WLS protein levels following VPS35 and MON2 suppression using RNAi. a–d Data presented as mean +/− s.e.m. from three independent biological replicates. Significance was determined using a one-way ANOVA with a post-hoc Dunnett’s test; *p < 0.05. e Representative western blot of endogenously expressed MIG-14::GFP (huSi2) protein levels in animals treated with control, vps-35, mon-2 or pad-1 RNAi. f Confocal imaging of MIG-14::GFP (green, huIs72) and the late endosomal and lysosomal marker LMP-1::mCherry (red, huEx149) in L1 larvae. The tail area, which includes the EGL-20 producing cells, is shown. Arrows indicate regions of colocalization. In all images, anterior is left and dorsal is up. Scale bar is 10 µm. g Staining of EGL-20::protA with rabbit-anti-goat-Alexa647 in L1 larvae. The EGL-20 producing cells are indicated with a solid line, the punctate gradient that is formed by EGL-20::protA is indicated with a dashed line. In all images, anterior is left and dorsal is up. Scale bar is 10 µm. h Systemic knock down of mon-2 or pad-1 interferes with the EGL-20/Wnt dependent posterior migration of the QL descendants (QL.d) in a vps-29(tm1320) sensitised genetic background. The percentage of animals with anteriorly displaced QL.d is shown (data are presented as mean +/− SD and include results from four independent biological replicates, with n ≥ 25 per replicate) *p = 1.158 × 10^–6 for vps-35 RNAi, *p = 1.44 × 10^–5 for mon-2 RNAi and *p = 0.0024 for pad-1 RNAi (Student’s t-test)