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. Author manuscript; available in PMC: 2018 Oct 3.
Published in final edited form as: Nature. 2018 Apr 3;556(7700):249–254. doi: 10.1038/s41586-018-0018-1

Figure 2. PFKFB4 phosphorylates SRC-3 by functioning as a protein kinase.

Figure 2

a, Upper panel-Recombinant GST-fused PFKFB4 incubated with full-length SRC-3 (SRC-3 FL) in presence of [32P]ATP in an in vitro kinase assay. Lower panels- SRC-3 and PFKFB4 protein levels were analyzed by immunoblotting. b, In vitro kinase assay of PFKFB4 in the presence of SRC-3 fragments expressing different domains or full length SRC-3-FL. c, HEK293T cells expressing Flag-tagged-SRC-3 and PFKFB4 cultured in different concentrations of glucose and immunoprecipitated by Flag or p-Ser/Thr antibodies followed by immunoblotting. d, MDA-MB-231 cells stably expressing shRNAs targeting PFKFB4 (sh-PFK#09 and sh-PFK#20) or control NT-shRNA grown in presence of 5mM, 25mM glucose or glucose withdrawn from cells grown in 25mM of glucose and replaced with 5mM (WD) for 6 hours. Protein levels of pSRC-3-S857, PFKFB4 and β-actin were detected by immunoblotting. e, HEK293T cells expressing pBIND, pBIND-SRC-3 or pBIND-SRC-3-S857A were transduced with Adv. GFP or PFKFB4, and cultured in 5mM or 25mM glucose followed by luciferase assay. [Boxes represent 25th to 75th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean, n=6 biologically independent experiments; Two-way ANOVA with Tukey’s Multiple comparisons test]. Data shown in (a–e) are representative of 3 biologically independent experiments with similar results. For exact P-values please refer to source data.