Co-expression of Hey1 strongly increases FBXO45 nuclear localization, but does not alter Hey repression capacity.

<p>(A) HeLa cells were transfected with FBXO45 expression vectors or its deletion mutants, either alone or together with peGFP-Hey1. Flag-tagged proteins were visualized with an anti-Flag antibody and an Alexa594 labeled secondary antibody. GFP fusion proteins were imaged directly and nuclei were stained with Hoechst33342 (B) The DNA-binding incompetent Hey1-RK₃ variant and the Hey1Δb mutant that lacks the nuclear localization signal were expressed together with FBXO45 and analyzed as before. (C) Quantification of predominant cytoplasmic or nuclear FBXO45 staining. 80 cells were counted per preparation. (D) FBXO45 does not influence Hey1 transcriptional activity. 293tet-FS-Hey1 cells stably transfected with a Hey1-luc (2,9 kb) reporter construct were induced with doxycycline (50 ng/ml and 500 ng/ml for 72 h) to induce FS-Hey1 expression. After 48 h the cells were transfected with increasing amounts of Flag-FBXO45 (100 ng and 200 ng) and tested after additional 24 h. Mean values and standard deviations of triplicates are shown.