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. 2018 Aug 17;8:12390. doi: 10.1038/s41598-018-29658-2

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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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Proteasome re-routing is beneficial to GFP-TDP-25 aggregation. (a) NSC34 overexpressing GFP-TDPs, and co-transfected with pCI-HA-Bag1 or pcDNA3. Left panel: NP-40 soluble extracts WB analysis (upper inset, 12% polyacrylamide gels), NP-40 soluble extracts FRA analysis (middle inset) and quantification of NP-40 soluble extracts FRA analysis (*p < 0.05 vs GFP-TDP-25 co-transfected with pcDNA3) (lower inset). Right panel: NP-40 insoluble extracts WB analysis (upper inset, 12% polyacrylamide gels), NP-40 insoluble extracts FRA analysis (middle inset) and quantification of NP-40 insoluble extracts FRA analysis (*p < 0.05 vs GFP-TDP-25 co-transfected with pcDNA3) (lower inset). (b) NSC34 overexpressing GFP-TDPs, and co-transfected with pCI-HA-Bag1 or pcDNA3 analysed with confocal microscope. 63X magnification. Green: GFP-TDPs; Red: hBag1; nuclei: DAPI. (c) C2C12 overexpressing GFP-TDPs, and co-transfected with pCI-HA-Bag1 or pcDNA3. Left panel: NP-40 soluble extracts WB analysis (upper inset, 12% polyacrylamide gels), NP-40 soluble extracts FRA analysis (middle inset) and quantification of NP-40 soluble extracts FRA analysis (**p < 0.05 vs GFP-TDP-35 co-transfected with pcDNA3) (lower inset). Right panel: NP-40 insoluble extracts WB analysis (upper inset, 12% polyacrylamide gels), NP-40 insoluble extracts FRA analysis (middle inset) and quantification of NP-40 insoluble extracts FRA analysis (*p < 0.05 vs GFP-TDP-25 co-transfected with pcDNA3). (d) C2C12 overexpressing GFP-TDPs, and co-transfected with pCI-HA-Bag1 or pcDNA3 as control vector analysed with confocal microscope. 63X magnification. Green: GFP-TDPs; Red: hBag1; nuclei: DAPI. (e) Microscope analysis of NSC34 transiently co-transfected with GFP-TDP-25 and HA-Bag1 or pcDNA3. Right panel: quantification of green fluorescence per cell (***p < 0.001 vs pcDNA3). Left panel: quantification of aggregates per cell. Small aggregates: dots composed of less than 1,000,000 pixels (***p < 0.001 vs pcDNA3). Big aggregates: dots composed of more than 1,000,000 pixels (**p < 0.01 vs pcDNA3). (f) Microscope analysis of C2C12 transiently co-transfected with GFP-TDP-25 and HA-Bag1 or pcDNA3. Right panel: quantification of green fluorescence per cell (**p < 0.01 vs pcDNA3). Left panel: quantification of aggregates per cell. Small aggregates: dots composed of less than 1,000,000 pixels. Big aggregates: dots composed of more than 1,000,000 pixels (*p < 0.05 vs pcDNA3). Full-length blots/gels are presented in Supplementary Figure S8.