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. 2023 Mar 10;14:1323. doi: 10.1038/s41467-023-37094-8

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© 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/.

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Fig. 4 — a Immunofluorescence analysis of the effect of PHGDH knockdown on PKM2 subcellular localization in HUVECs. Blue: DAPI; Red: PKM2. b Effect of PHGDH knockdown on the amount of PKM2, PSAT1 and PSPH localized in the cytoplasm (Cyto) and nucleus (Nuc) of HUVECs as determined by subcellular fractionation analysis. Effect of PHGDH knockdown (shPHGDH) on PKM2 K433ac in HUVECs as determined by immunoblots (c) and immunofluorescence (d). More lysates from PHGDH-knockdown HUVECs were loaded to keep PKM2 at similar levels. Effect of PHGDH overexpression (e) and PHGDH knockdown (f) on the interaction between PKM2 and p300 in HUVECs as determined by Co-IP. To ensure equal loading of PKM2, less cell lysate from PHGDH-overexpression (PHGDH OE) HUVECs (e) and more lysate from PHGDH-knockdown (shPHGDH) HUVECs (f) were used for immunoprecipitation. Actin and histone H3 were used as loading controls. g Purified recombinant PHGDH increased the direct binding of PKM2 with p300 as determined by in vitro Co-IP. p300 was immobilized on agarose beads from HeLa cells with anti-p300 and then incubated with purified recombinant PKM2 in the presence of increasing amount of purified recombinant PHGDH. h Molecular docking showing the formation of PHGDH/PKM2/p300. i PKM2 K433R decreased its interaction with PHGDH as determined by Co-IP. HUVECs were individually infected with lentiviruses to stably express WT FLAG-PKM2, FLAG-PKM2 K433R and FLAG-PKM2 K433Q. FLAG-PKM2 was IPed with anti-FLAG antibody. j In vitro Co-IP showing the recombinant purified His-PKM2 K433R mutant had reduced interaction with PHGDH (Myc-PHGDH) when compared with His-PKM2 K433Q. k p300 knockdown (sip300) decreased the binding of PKM2 with PHGDH in HUVECs. l Effect of ectopic expression of WT PKM2, PKM2 K433R and PKM2 K433Q on cellular senescence. HUVECs were infected with lentiviruses to stably express WT FLAG-PKM2, FLAG-PKM2 K433R and FLAG-PKM2 K433Q. For a–f, k, l, HUVECs (P10) were used in the experiments. For c, data represent means ± SE; n = 3 independent experiments. For l, data represent means ± SE; n = 5 independent experiments. Two-sided t-tests were used for statistical analysis. For a, b, d–g, i–k, a typical example of three biological replicates is shown.