Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2023 Apr 19;14:2150. doi: 10.1038/s41467-023-37464-2

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2023

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/.

PMC Copyright notice

Fig. 3 — A Validation of PPI disruption using co-immunoprecipitation assays. Flag-CHMP2B was immunoprecipitated in the presence of HA-tagged A53T a-syn and GFP-tagged peptides. Negative controls were GFP alone (CTL) and a GFP-tagged scrambled version of PDpep1.3 (Scramble1.3); neither markedly co-immunoprecipitated with CHMP2B nor disrupted the interaction between CHMP2B and A53T a-syn. B Fluorescence polarization (FP) binding assay of FITC-labeled PDpep1.3 (blue) and displacement of the fluorescent peptide with increasing concentrations of a-syn (black). Error bars represent ± s.d. of the fit (n = 3) (left panel). FP binding assay of FITC-labeled a-syn peptides. Error bars represent ± s.d. of the fit (n = 3) (right panel). C PDpep1.3 restores reduced LAMP1 expression due to A53T a-syn in HEK293 cells as shown by confocal micrographs (scale bars = 15 μm). Representative immunoblots of D LAMP1 levels or E CD63 levels in HEK293 cells upon co-transfection of A53T a-syn and peptides. Controls were GFP alone (pLJM1) and a GFP-tagged scrambled version of the initial peptide (scramble). Loading control was beta-actin. Experiments were done in triplicate. F Endolysosomal flux assay using flow cytometry in HEK293 cells co-transfected with A53T a-syn or control vector plus Scramble1.3 or PDpep1.3. Cells were treated with the lysosomal inhibitor Leupeptin (Leu) as indicated. Data represent means ± s.d. (unpaired two-tailed t-test, t(4) = 2.776; A53T+PDpep1.3, P = 0.0010; n = 3). G Autophagy flux assay in HEK293 cells stably expressing a luminescent LC3-HiBiT reporter co-transfected with A53T a-syn or empty vector (EV) plus Flag-Scramble1.3 or Flag-PDpep1.3. Cells were treated with the lysosomal inhibitor Bafilomycin A1 or the autophagy inducer Rapamycin as indicated. Bars represent means ± s.e.m. (n = 3). H Representative images of cortical neurons transduced with A53T a-syn or EV plus Scramble1.3-RFP or PDpep1.3-RFP (scale bars = 5 μm). I Quantification of LAMP1 fluorescence in RFP-positive neurons. Bars represent means ± s.e.m. (nested one-way ANOVA, F(3, 102) = 4.430; P = 0.0057; n = 3). J The proposed mechanism of PDpep1.3 is that the peptide disrupts the a-syn-CHMP2B interaction to restore degradation of a-syn by the endolysosomal pathway (created using Servier Medical Art at https://smart.servier.com/). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.