Fig. 5: TRIM55 interacts with and ubiquitinates NF90.

A The cells transfected with GFP-tagged TRIM55 were immunostained for NF90 (red), and DNA (DAPI, blue). Representative fluorescence images of NF90 and TRIM55 localization in cells are shown in the figure. There is space for co-positioning between NF90 and TRIM55. B Co-IP assay was performed in HEK-293T cells transfected with TRIM55-GFP and NF90-Flag to explore the interaction between exogenous TRIM55 and NF90. C Co-IP experiments were conducted in HCC cells with TRIM55 overexpression to explore the interaction between exogenous TRIM55 and endogenous NF90. D Endogenous co-IP experiments indicated that TRIM55 interacted with NF90 in Huh7 and SK-Hep-1 cells. IgG was used as a negative antibody. E NF90 expression levels in control and TRIM55 overexpressing HCC cells were determined via western blotting. F IHC analysis of the expression of NF90 proteins in control and TRIM55-overexpressed xenografts. G Control and TRIM55 overexpressing HCC cells were treated with CHX (200 μg/ml) for time intervals indicated. The cell lysates were subjected to immunoblotting (left panel) and NF90 expression level was quantified (right panel). H Control and TRIM55 overexpressing HCC cells were treated with MG132 (10 µM) for 8 h. Then, the NF90 was examined using western blotting. I Control and TRIM55 overexpressing HCC cells were treated with MG132 10 µM for 8 h. Cell lysates were then subjected to immunoprecipitation with an anti-NF90 antibody. The resulting immunocomplexes were immunoblotted with anti-NF90 and anti-ubiquitin antibodies. J HEK293T cells overexpressing FLAG-NF90, GFP-TRIM55, and HA-tagged different forms of ubiquitin were treated with MG132 (10 μM) for 8 h. The cell lysates were immunoprecipitated by using anti-FLAG M2 affinity gel. The ubiquitination levels of NF90 were detected using anti-HA antibody. n = 3, **P < 0.01, ***P < 0.001.