Fig. 2: PGK1 Kcr at K131, K156, and K220 sites decrease under hypoxia.

a, b The MS/MS spectrum (a) and extracted ion chromatograms from LC-MS/MS analysis (b) of the in vivo derived PGK K131cr peptide, the synthetic standard PGK K131cr peptide, and their mixture. The b ion refers to the N-terminal parts of the peptide, and the y ion refers to the C-terminal parts of the peptide. c The ratio and p value (unpaired two-tailed Student’s t test) of PGK1 Kcr on different sites under hypoxia versus normoxia. d IP assays were performed with anti-Flag M2 beads using HEK293T cells expressing Flag-tagged PGK1 WT, K131R, K156R, K220R, or 3KR, followed by immunoblotting with pan anti-Kcr and anti-Flag antibodies. e Sequence alignment of the PGK1 amino acid residues across various species. f Dot blot assays were performed to verify the specificity of PGK1 K131cr antibody against PGK1 K131cr, K156cr, and K220cr. g IP assays were performed with anti-Flag M2 beads using HEK293T cells expressing Flag-tagged PGK1 K131R, followed by immunoblotting with the specific antibody against PGK1 K131cr. h MDA-MB231 cells were cultured under the stimulation of normoxia or hypoxia for 12 h and subjected to IP assays using anti-PGK1 or anti-IgG antibodies, followed by immunoblotting with the specific antibody against PGK1 K131cr. i MDA-MB231 cells were incubated with or without 10 mM sodium crotonate (pH 7.4) and subjected to IP assays using the indicated antibodies, followed by immunoblotting with the specific antibody against PGK1 K131cr. d, f–i Data were verified in at least three independent experiments. Source data are provided as a Source Data file.