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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. 3 — a Mass spectrometry analysis of PHGDH- and PKM2-interaction proteins. The ectopic expressed Myc-PHGDH and FLAG-PKM2 were immunoprecipitated from HeLa cells with anti-Myc and anti-FLAG antibodies as the bait. b Co-IP assay showing PKM2 interacted with PHGDH, PSAT1 and PSPH. PKM2 and PHGDH were immunoprecipitated from HUVEC cells by anti-PKM2 and anti-PHGDH antibodies, respectively. c In vitro GST pull-down assay showing the direct interaction between purified recombinant GST-PHGDH and His-PKM2. d Immunoblots of PKM2 in PHGDH-knockdown (shPHGDH) and PHGDH overexpression (PHGDH OE) HUVECs. e PHGDH knockdown impaired PKM2 stability. Control (shCtrl) and PHGDH-knockdown (shPHGDH) HUVECs were treated with cycloheximide (CHX) (50 μg/ml) for 0–8 h. f Immunoblots of PHGDH and PKM2 in control (shCtrl) and ATF4-knockdown (siATF4) HUVECs. g Effect of autophagy inhibitor, leupeptin on PKM2 levels in control (shCtrl) and PHGDH-knockdown (shPHGDH) HUVECs. shCtrl and shPHGDH cells were treated with 20 μM leupeptin for 6 h. h Molecular docking showing the interaction between PKM2 and PHGDH. PKM2 K305 was indicated by an arrow. i Immunoblots of PKM2 K305ac in control (shCtrl) and PHGDH-knockdown (shPHGDH) HUVECs. j Co-IP assay showing overexpression of PHGDH (PHGDH OE) reduced the interaction between PKM2 and PCAF. Asterisk indicates non-specific bands. k In vitro Co-IP showing recombinant purified PHGDH decreased the direct binding of PKM2 with PCAF. FLAG-PCAF was purified from HeLa cells and incubated with purified recombinant His-PKM2 in the presence of increasing amount of purified recombinant PHGDH, which were then immunoprecipitated with anti-FLAG beads. l PKM2 K305R mutant was more stable than WT PKM2 in PHGDH-knockdown (siPHGDH) cells. HeLa cells that stably express WT FLAG-PKM2 and FLAG-PKM2 K305R were transfected with siPHGDH and then treated with CHX. m Effect of ectopic expression of WT PKM2 and PKM2 K305R mutant on cellular senescence. HUVECs that stably express FLAG-WT PKM2 and FLAG-PKM2 K305R were transfected with siPKM2 to knock down the endogenous PKM2. For e, l, data represent means ± SE; n = 3 independent experiments. For m, n = 4 independent experiments. Two-sided t-tests were used for statistical analysis. For b–d, f, g, i–k, a typical example of at least two biological replicates is shown.