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. 2022 Nov 24;13:7215. doi: 10.1038/s41467-022-34747-y

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© The Author(s) 2022

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. 2 — a The Ser79 phosphorylation modification of PROX1 peptides identified through liquid chromatography-tandem mass spectrometry. b The substrate motif of AMPK kinases is shown (lower), and the Ser79 site of PROX1 is conserved in vertebrate. c Coomassie blue staining of GST and GST-AMPKα2 incubated with in vitro translated PROX1, PROX1 was detected by anti-PROX1 antibody after GST-pulldown. d Endogenous PROX1 and AMPKα2 in the Huh7 cells were visualized under fluorescent microscopy (n = 3 independent experiments). Nuclei were stained with DAPI. Scale bar, 10 µm. e Western blot analysis Huh7 cell lysates as indicated. f The domain organization of PROX1 and the deletion constructs. PD1, prospero domain1; HD, homeodomain; PD2, prospero domain 2. g Input, coomassie blue staining of each GST-PROX1 fragment incubated with in vitro translated AMPKα2. AMPKα2 was detected by anti-AMPKα2 antibody after GST-pulldown. h The P1-WT and P1-S79A of PROX1 as indicated was detected using a phosphor-specific antibody against Ser79 of PROX1. i HEK293T-expressed wide type (WT) and replacement of S79 with Ala (A) or Glu (E) in the FLAG-PROX1 (S79A and S79E) incubated with GST-AMPKα2. j Western blot analysis HEK293T cell lysates as indicated. k Immunoblot analysis of HEK293T cell lysates transfected with the indicated PROX1 and HA-AMPKα2 plasmids. l Immunoblot analysis of the FLAG-IP and cell lysates from transfected with the indicated constructs. m Immunoblot analysis of MEFs cell lysates as indicated. n Representative IHC staining images and statistical data of the murine lung tumour tissues from Kras^G12D (K), Kras^G12D/Lkb1^L/L (KL) and Kras^G12D-sgAmpk (KA) mouse (n = 5). Scale bar, 50 µm. o Representative IHC staining images and statistical data of the liver tissues from normal, fasted and metformin (500 mg/L) treatment mice (n = 6). Scale bar, 50 µm. p, q Representative western blot (p) and the corresponding quantified graph (q) of Huh7 cell lysates are shown. n = 3 independent experiments. IB, immunoblot; IP, immunoprecipitation. The immunoblots are repeated independently with similar results at three times. For n, o and q, data represent the mean ± SD. Statistical significance was assessed using two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.