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. 2023 Jul 25;14(7):463. doi: 10.1038/s41419-023-05998-4

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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. 2 — A Immuno-precipitates of GFP or GFP-GSTP1 analyzed by Western blots. B Endogenous G6PD protein is also Tyr phosphorylated. MCF-7 cells were seeded in 8 ten-cm dishes and cultured for two days. Cell lysates from 4 dishes were immunoprecipitated with control IgG, the rest with anti-G6PD antibodies. C Sequence alignment of phosphorylated Tyr residues among species. D Nitrocellulose membranes were spotted with indicated amounts of indicated peptides and probed with α-pG6PD Y249 and α-pG6PD Y322. E Flag-tagged WT and mutants were immunopurified with flag affinity (M2) beads and Western-blotted with α-pG6PD Y249 and α-pG6PD Y322 antibodies. F Tyr phosphorylation levels of G6PD WT vs. mutants were determined. G MCF-7 cells were treated with indicated Tyr kinase inhibitors for 12 h and Flag-tagged G6PD immunopurified to test phosphorylation levels. H HA-SRC (active) or HA-KM (kinase inactive SRC) were co-expressed with Flag-tagged G6PD in MCF-7 cells for 24 h, and Flag-tagged G6PD was immunopurified and immunoblotted with antibodies. I HA-SRC or HA-tag control empty plasmids were co-transfected with plasmids expressing WT (G6PD) and mutants (2YF/2YA) for 24 h, and Flag-tagged G6PD was immunopurified and immunoblotted with antibodies. J Two siRNA (#1 and #2) targeting SRC were transfected into MCF-7 cells. After 24 h, Flag-G6PD was also transfected for another 24 h. K Recombinant WT (G6PD) was expressed and purified from E. coil and incubated with or without recombinant SRC (active) and ATP. The mixture was subjected to analysis detecting phosphorylation of Y249 and Y322 (upper panel), and for Coomassie Brilliant Blue (CBB) staining that normalized input proteins (lower panel). L Recombinant WT (G6PD) and mutants (2YF/2YA) were incubated with recombinant SRC (active) and ATP. The mixture was subjected to analysis detecting phosphorylation at Y249 and Y322 (upper panel) and for CBB staining (lower panel) that normalized input proteins.