Fig. 4.

UXT-V2 overexpression increases increases Fbxo7 levels levels into the cell nucleus. A and B) U2OS cells were transfected with the indicated plasmids, and the cytosolic- and nuclear-enriched fractions were resolved by SDS-PAGE and analyzed by western blotting with the indicated antibodies. Histone and Akt were probed as nuclear and cytoplasmic markers, respectively. GraphPad Prism was used to statistical test (Student's t-test), and all assays were performed in triplicate. **: p ≤ 0.05 and ***: p ≤ 0.01. C) U2OS cells were transfected with (i) Fbxo7, (ii) Fbxo7 and UXT-V1-M13G or (iii) Fbxo7 and UXT—V2. The slides were incubated with anti-HA or anti-Fbxo7 and visualized by goat anti-rabbit-Alexa 488 or donkey anti-mouse-Alexa 594. Nuclei were stained with DAPI and an objective of 63× was used. D) HEK293T cells were transfected with pEGFP-N1 or UXT-V2-HA, and total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. The Fbxo7 protein levels are presented in the graph with densitometry values obtained by ImageJ. E) mRNA was purified from HEK293T cells transfected with pcDNA3, UXT-V2-HA or pEEGFP-N1. RT-qPCR analysis was carried out for FBXO7 expression and evaluated in comparison to the GAPDH. No significant differences were found among the groups (n = 3) by using one-way ANOVA (p value = 0.4807).