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. 2023 Feb 28;14:1143. doi: 10.1038/s41467-023-36693-9

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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. 5 — A Sites of CIP2A-B56α cross-links (in red; PDB: 6NTS) mapped in relation to indicated LxxIxE-binding groove of B56α. Residues indicated in light purple constitutes the LxxIxE-binding region of B56α between amino acids 212-271. Residues in dark purple indicate amino acids involved in PP2A-A interaction as explained in Fig. 4. B CIP2A(1-560)V5 out-competes prototypical LxxIxE motif target BubR1(LxxIxE peptide 647-720) from its direct association with B56α. Source data are provided as a Source Data file. C Quantification of GST pull-down data from (B) shown as a mean + S.E.M from N = 3 biological repeats. Two-sided t-test. D B56α competition assay using GST-BubR1(647-720) alone, or in combination with CIP2A N-terminal head peptide (aa. 18-40). Source data are provided as a Source Data file. E Quantification for data from D shown as a mean + S.E.M from N = 4 biological repeats. Two-sided t-test. F In vitro binding assay using purified recombinant GST-tagged CIP2A(1-560) and B56α WT or B56 variants Y215Q and R222E, which contain mutations that prevent interaction with LxxIxE groove substrate proteins¹⁰. Source data are provided as a Source Data file. G Quantification of data from (F) shown as mean + S.E.M from N = 3 biological repeats. H GFP-tagged B56α WT or indicated triple mutant were expressed in HEK-293-T cells. The amount of PP2A-A subunit bound to GFP-tagged B56α upon GFP trap pull-down was quantified by anti-PP2A-A immunoblotting. Shown are the mean values + S.E.M of the ratios of the quantified anti-PP2A-A signal versus the quantified anti-GFP signal, relative to the B56α WT (set at 100 %) from N = 3 biological replicates. A two-sided one-sample t-test. I Triple B56α mutant (K181A/K217A/K227A) exhibits loss of K227 (B56)-D117(PP2A-A) salt bridge. Overlay of PP2A-B56α (PDBID 6NTS: PP2A-A, yellow; B56α, beige) and PP2A-B56γ (PDBID 2IAE: PP2A-a, light cyan, B56γ, cyan), with PP2A-A D177 and B56α/B56γ K227/K202 residues shown as sticks and labelled. H-bond interactions are shown using dotted lines. Image was generated using Pymol.