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. 2020 Oct 27;10(11):783. doi: 10.3390/brainsci10110783

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Impaired endosome-to-Golgi retrieval of cation-independent mannose-6-phosphate receptor (CI-MPR) in A320V-expressing cells. (A) Human embryonic kidney (HEK)-293T cells were transiently cotransfected with wild-type (WT) or mutant (A320V) VPS35-ZsYellow1 and α-synuclein (SNCA)-Myc-His cDNAs. The levels of exogenously expressed VPS35-ZsYellow1 and SNCA and endogenous CI-MPR, cathepsin D (CTSD), and lysosomal-associated membrane protein 2 (LAMP2A) were examined using Western blot analysis. Representative immunoblots from three independent experiments are shown. The densitometric quantification of CI-MPR, CTSD, SNCA, and LAMP2A versus β-actin (ACTB) is presented in the right panel. Data are expressed as the mean ± standard deviation. (B) Expression of VPS35 and CI-MPR proteins was examined by confocal microscopy and colocalization analysis. Wild-type or mutant VPS35 was labeled with green, while CI-MPR was stained with red by Cy5 fluorescence. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI; blue). The graph below represent quantification of colocalization with the means of three independent experiments.

Impaired endosome-to-Golgi retrieval of cation-independent mannose-6-phosphate receptor (CI-MPR) in A320V-expressing cells. (A) Human embryonic kidney (HEK)-293T cells were transiently cotransfected with wild-type (WT) or mutant (A320V) VPS35-ZsYellow1 and α-synuclein (SNCA)-Myc-His cDNAs. The levels of exogenously expressed VPS35-ZsYellow1 and SNCA and endogenous CI-MPR, cathepsin D (CTSD), and lysosomal-associated membrane protein 2 (LAMP2A) were examined using Western blot analysis. Representative immunoblots from three independent experiments are shown. The densitometric quantification of CI-MPR, CTSD, SNCA, and LAMP2A versus β-actin (ACTB) is presented in the right panel. Data are expressed as the mean ± standard deviation. (B) Expression of VPS35 and CI-MPR proteins was examined by confocal microscopy and colocalization analysis. Wild-type or mutant VPS35 was labeled with green, while CI-MPR was stained with red by Cy5 fluorescence. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI; blue). The graph below represent quantification of colocalization with the means of three independent experiments.