Fig. 6: TIP30 inhibits importin β-dependent nuclear import of UBE3A.

A Scheme of an experimental pipeline used to study the role of TIP30 in the subcellular distribution of UBE3A. U2OS cells were transfected with one of the following expression plasmids: m(mouse)UBE3A-Iso3, mUBE3A-Iso2, and r(at)TIP30, or a combination of mUBE3A-Iso3:rTIP30 or mUBE3A-Iso2:rTIP30 in a ratio of 1:1 or 1:3 (B–D). HEK293T cells were transfected with Tip30 siRNA or negative control siRNA (n.c.), and treated with importazole (importin β-inhibitor, Impβ^INH, 20 µM), or vehicle (E–J). B–D Representative images (B) and quantification of UBE3A⁺ %area (C) and intensity (D) in the nucleus:cytoplasm of the cells (n = 3 samples). Scale bar 15 µm. E,F Representative images (E) and quantification (F) of UBE3A⁺ %area in the nucleus:cytoplasm of the cells (n = 5 samples). Scale bar 30 µm. G,H Representative images (G) and quantification (H) of ABCA1⁺ %Area (n = 3 samples). Scale bar 30 µm. I ABCA1 protein level determined with immunoblotting (n = 5 samples). ABCA1 protein levels are normalized to Cy5 levels (loading control). J Relative lipid load determined with BODIPY FACS [n(n.c.) = 11 samples; n(Tip30) = 13 samples)]. Data were collected from ≥3 independent experiments, and all data were represented as mean ± SEM. Statistical significance was determined by two-sided Student’s t-test (H–J) or two-sided one-way ANOVA (C, D, F). *P < 0.05, **P < 0.01, and ***P < 0.001. If p values are not indicated, the result was not significant. Created in BioRender. Bogie, J. (2025) https://BioRender.com/2yr70n3.