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. Author manuscript; available in PMC: 2018 Feb 7.
Published in final edited form as: Nat Chem Biol. 2017 Aug 7;13(10):1081–1087. doi: 10.1038/nchembio.2453

Figure 3. 1,3-BPG is an alternative source of PGAM1 phosphorylation.

Figure 3

a) Western blot analysis of PGAM1 overexpression lysates from wt and BPGM knockout HEK 293T cell lysates using an α–pHis and α–PGAM1 antibody (α–actin antibody was used as a loading control). b) Western blot analysis of a PGAM1 phosphorylation time course in the presence of 2,3-BPG or 1,3-BPG (membranes were stained with Coomassie to serve as a loading control). c) 1,3-BPG-treated PGAM1 was subjected to trypsinization and LC-MS analysis. Shown is the MS/MS spectrum of the tryptic peptide of PGAM1 containing the pHis site (His-11) and in mirror image is shown the MS/MS spectrum of 2,3-BPG induced phosphorylation of PGAM1 to demonstrate that both 1,3-BPG and 2,3-BPG result in His-11 phosphorylation. d) LC-MS analysis of PGAM1 phosphorylation with 1,3-BPG in the absence of 2-PG, low 2-PG (50 μM), or high 2-PG (1000 μM). Extracted ion chromatograms for 1,3-BPG and 2,3-BPG (m/z=264.952) are shown for each of the described reaction conditions. See Supplementary Figure 24 for full Western blot images.