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. 2017 Jan 5;8(1):e2525. doi: 10.1038/cddis.2016.424

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Copyright © 2017 The Author(s)

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a and b). Prdx6-deficient LECs displayed insignificantly low levels of Phospholipase A₂ as well as lower GSH peroxidase activities compared with Prdx6^+/+. Prdx6^+/+ and Prdx6^−/− LECs cultured in identical conditions as described in 'Materials and methods'. Cells were harvested and total extracts containing equal amounts of proteins were processed to measure PLA₂ (a) and glutathione peroxidase activity (b) following the company's protocols. Black bars show significantly reduced PLA₂ and GSH peroxidase activities in Prdx6-deficient cells. The data represent the mean±SD from three independent experiments (*P<0.001). Upper panel, a schematic illustration of active sites responsible for PLA₂ (S32/H26/D140) and GSH peroxidase (C47) activities. (c and d). Disruption of Sumoylation motif K122/142 R in Prdx6 protein promoted PLA₂ and glutathione peroxidase activities. Prdx6^−/−LECs were transfected with pEGFP-Vector, pGFP-Prdx6WT and its mutants, pGFP-Prdx6K122R, pGFP-Prdx6K142 R and pGFP-Prdx6K122/142R fused to GFP plasmids. After 48 h, total lysates containing equal amounts of proteins were processed for PLA₂ (c) and GSH peroxidase (d) activities through Enzchek PLA₂ and GSH peroxidase assay kits (Invitrogen), respectively. (Prdx6WT versus mutants; *P<0.001; **P<0.05). (e and f). Recombinant mutant Prdx6K122/142 R protein had increased PLA₂ and GSH peroxidase activities compared with Prdx6WT. Prdx6^−/−LECs were transduced with TAT-HA-Prdx6 and its mutant TAT-HA-Prdx6K122/142 R. After 24 h, total protein was isolated and assays were performed for PLA₂ (e) and Glutathione peroxidase (f) activities as described in ‘Materials and methods' section. The data represent the mean±SD from three independent experiments (*P<0.001). (g and h). Sumo1 overexpression diminished phospholipase PLA₂ and GSH peroxidase activities. Prdx6^−/−LECs were co-expressed with pEGFP-Sumo1 along with pEGFP-Vector or pGFP-Prdx6WT or its mutant K122/142 R, 48 h later total cell lysates containing equal amounts of proteins were utilized to measure PLA₂ (g) and glutathione peroxidase (h) activities; results are presented as nmol/min/mg protein and units/min/mg protein, respectively. The data represent the mean±SD from three independent experiments (*P<0.001)