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. 2020 Sep 28;9(9):85. doi: 10.1038/s41389-020-00271-1

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© The Author(s) 2020

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. 3 — a Western blotting of exogenous DHPS phosphorylation in HEK293A cells. HEK293A cells expressing SFB-tagged DHPS were exposed to DMSO or different dosages of PMA for 30 min. Cell lysates were pulled down with S protein beads, and the indicated proteins were detected by Western blotting. Relative levels of pS/T signals after normalization to the flag signals were shown. Data represent a representative experiment from three independent experiments. b In vitro kinase assay of DHPS. Purified GST or GST-DHPS was incubated with or without ERK. γ-³²P signals were detected by PhosphorImager. The proteins were detected by Coomassie Blue staining. Arrows mark the radiative signals of GST-DHPS phosphorylation and ERK auto-phosphorylation. c Sequence alignment indicates two possible ERK phosphorylation sites (S/T-P) that are highly conserved from yeast to humans. d In vitro kinase assay of DHPS variants. Purified GST-DHPS-WT or variants (T202A, S233A, 2A/T202A-S233A) were incubated with or without ERK. γ-³²P signals were detected by PhosphorImager. The proteins were detected by Coomassie Blue staining. Arrows marked the radiative signals of GST-DHPS phosphorylation and ERK self-phosphorylation. Data represent a representative experiment from three independent experiments. e Surface of the human DHPS-dimer crystal structure (PDB: 1ROZ). The substrates are marked as green (upper: NADPH, lower: spermidine). Ser-233 (S233) is marked with yellow and is located in the NADPH binding pocket. The other essential amino acid, Asp-238 (D238), is also marked with yellow. The EBM described in this study is circled and marked with yellow. A close-up of the surface, including the Ser-233 site, is shown in the right panel. f Comparison of enzymatic activities of DHPS variants. The enzymatic activity assays were performed with purified GST-eIF5A (1 μg proteins in each sample) and different GST-DHPS variants (0.2 μg proteins in each sample). The proteins were detected by Coomassie Blue staining. Arrows mark the proteins of GST-DHPS variants and GST-eIF5A. The activities of the mutant enzymes are expressed as percent of the wild-type enzyme in each group. Results were presented as means ± S.D. from three independent experiments. ***, p < 0.001. *, p < 0.05. P values were obtained using Student’s t test.