Fig. 1: The global crotonylome reveals downregulation of PGK1 crotonylation under hypoxia. | Nature Communications

Fig. 1: The global crotonylome reveals downregulation of PGK1 crotonylation under hypoxia.

From: Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle

Fig. 1

a, b Schematic illustration of TMT quantitative proteomics workflow to determine the levels of Kcr proteins in MDA-MB231 cells cultured under normoxic or hypoxic conditions for 12 h (a). Each group had 3 biologically independent samples. The relative levels of glucose consumption and lactate production were measured to confirm the effectiveness of hypoxia treatment (b). Data are shown as the mean ± SD, n = 3 biologically independent samples (unpaired two-tailed Student’s t-test). c Column chart showing the statistical proteins, peptides and sites information of TMT quantitative proteomics results. d Identified differential Kcr sites and proteins were counted. e Pie graph showing the subcellular distribution of identified Kcr differential proteins. f KEGG pathway enrichment of identified Kcr differential proteins. The dot size and color represent the protein number and the p value (unpaired two-tailed Student’s t-test) of the corresponding pathway, respectively. g Volcano plots showing the quantification and p value (unpaired two-tailed Student’s t test) of identified differential Kcr sites. h MDA-MB231 cells were cultured under normoxia or hypoxia for 12 h and subjected to IP assays using anti-PGK1 or anti-IgG antibodies, followed by immunoblotting with pan anti-Kcr and anti-PGK1 antibodies. WCL, whole-cell lysate. i IP assays were performed with anti-Flag M2 beads using HEK293T cells expressing the Flag-tagged PGK1 cultured under normoxia or hypoxia for 12 h, followed by immunoblotting with pan anti-Kcr and anti-Flag antibodies. j HEK293T cells expressing Flag-tagged PGK1 were pretreated with 2.5, 5, or 10 mM sodium crotonate (pH 7.4) or 10 mM sodium acetate (pH 7.4) for 12 h. IP assays were performed with anti-Flag M2 beads, followed by immunoblotting with the indicated antibodies. k HEK293T cells expressing Flag-tagged PGK1 were pretreated with or without 10 mM sodium crotonate (pH 7.4) and subsequently cultured under normoxia or hypoxia for 12 h. IP assays were performed with anti-Flag M2 beads, followed by immunoblotting with the indicated antibodies. b, h–k Data were verified in at least three independent experiments. Source data are provided as a Source Data file.

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