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. 2017 Sep 29;7:12456. doi: 10.1038/s41598-017-12334-2

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© The Author(s) 2017

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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GSK3β phosphorylates FE65 T579 (A) Alignment of FE65 PTB2 sequences from various species. T597 is bolded (reference to human). Secondary structures of human FE65 PTB2 are indicated according to^18,19. The position of β^ct is also shown. (B) FE65 and FE65 T579A were transfected to HEK293 cells together with or without GSK3β. Cell lysates were resolved on 8% (top panel) and 12% (second panel) SDS-PAGE gels for determination of FE65 migration patterns and total FE65 levels, respectively. Anti-FE65 E20 antibody was used to detect FE65. Expression of HA-tagged GSK3β was confirmed by immunoblotting using anti-HA 12CA5 antibody. α-tubulin was detected by anti-α-tubulin DM1A antibody as loading control. UT, untransfected. The relative amount of phosphorylated-FE65 was analyzed by densitometry (LI-COR® Image Studio™ Software). n = 4. *P < 0.001. Results are means ± S.D. (C) Bacterially expressed His-tagged FE65 PTB2 (531–666) wild-type/T579A were incubated with HA-tagged GSK3β immunoprecipitated from transfected cell lysate together with [γ-³²P]-ATP for an hour at 30 °C. Arrow denotes the specific phosphorylated band in lane 4. Lower panel shows the Coomassie Blue staining. RM: reaction mixture only without substrate. WT, His-tagged FE65 PTB2 (531–666) wild-type. T579A, His-tagged FE65 PTB2 (531–666) T579A mutant. (D) FE65 and FE65 T579A were transfected to HEK293 cells together with or without GSK3β. T579 phosphorylated FE65 was detected by pT579 FE65 phospho-specific antibody. UT indicates untransfected. FE65, GSK3β and α-tubulin were detected by immunoblotting. The relative amount of p579 FE65 level was analyzed by densitometry. n = 6. *P < 0.001. Results are means ± S.D. (E) Endogenous FE65 was immunoprecipitated from adult rat brain lysate by using anti-FE65 E20. FE65 in the brain lysate and immunoprecipitate were revealed by immunoblotting. T579 phosphorylated FE65 was detected with pT579 FE65 antibody. Arrow denotes T579 phosphorylated FE65.