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. 2017 Apr 25;31(11):2491–2502. doi: 10.1038/leu.2017.105

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Copyright © 2017 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

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Both AE9a and AE9aNT interact with the Notch-corepressor SHARP but only AE9a derepresses Notch target genes. (a) AE9a and AE9aNT interact with the SPOC domain of SHARP. GST pulldown assay was performed by using bacterially purified GST-SPOC or GST only as control and cell free synthesized AE9a, AE9aNT or ETO as positive control. (b) AE9a but not AE9aNT derepresses RBP-J-dependent transcription. Activity of AE9a and AE9aNT was tested in HeLa cells by luciferase assays using the RBP-dependent reporter construct pGA891/6 and RBP or RBP-SPOC fusion proteins together with ETO, AE9a or AE9aNT. Mean values ± SD (error bars) based on at least three independent experiments are shown (^nsNot significant, ***P<0.001, unpaired Student’s t-test). (c) Surface view of the human SPOC domain of SHARP (PDB1OW1). The four amino acids R3554 (red), R3552 (orange), Y3602 (dark red) and K3516 (magenta), which were mutated to alanines are shown. Approximately 90° views are shown. (d) SPOC domain mutations show decreased interaction with AE9a (upper panel, lanes 3, 4 and 5). HEK-293 cells were transfected with the indicated expression constructs for Flag-tagged wt SPOC domain [Flag-SPOC(wt)] or mutant SPOC domains [Flag-SPOC (Y3602A), Flag-SPOC(K3516A) and Flag-SPOC(R3552A/R3554A)] together with an AE9a-GFP expression construct. Expression was verified by western blotting for AE9a (middle panel, lanes 2, 3, 4, 5), SPOC(wt) (lower panel, lane 2) and SPOC mutants (lower panel, lanes 3, 4 and 5). (e) Mutations of the SPOC domain of SHARP compromise the derepressing activity of AE9a. Activity of AE9a was tested in luciferase assays using the RBPJ-dependent reporter construct pGA891/6 and RBPJ-SPOC fusion proteins together with AE9a. The RBP-SPOC mutants show decreased AE9a mediated derepression of transcription. Mean values ± SD (error bars) based on four independent experiments are shown (^nsNot significant, *P<0.05, **P<0.01, ***P<0.001, unpaired Student’s t-test). (f) The AE9a/CBFβ interaction is required to dysregulate Notch target genes (left panel) but not AML1 (right panel) target genes in HoxB4 cells. Total RNA from HoxB4 cells transfected with empty vector (control), AE9a or AE9aNT was reverse transcribed in cDNA and analyzed via qPCR. Data were normalized to the housekeeping gene TATA-binding protein. The mean ± SD of triplicate experiments is shown (^nsNot significant, *P<0.05, **P<0.01, ***P<0.001, unpaired Student’s t-test).