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. 2022 Apr 21;12:843157. doi: 10.3389/fonc.2022.843157

Figure 2.

Figure 2

Effects of Tip110 intracellular localization and ubiquitination on NF-κB activity. (A) Schematic of Tip110 and its respective deletion mutants. Tip110 consists of seven half-a-tetratricopeptide repeats (HAT), a nuclear localization signal (NLS), and two RNA recognition motifs (RRM). (B, C) 293T were transfected with 0.6 µg pGL3-NF-κB(3)-Luc and Tip110.His or one of the Tip110 deletion mutants. Cells were harvested 48 hr post-transfection for the luciferase reporter gene assay (B) and Western blotting for Tip110 and its mutant expression using a monoclonal (M) and polyclonal (R) anti-Tip110 antibody (C). (D) 293T were transfected with UB.HA and Tip110.His, or Tip110ΔNLS, with and without USP15.Myc cultured for 48 hr, treated with MG132 (10 µM) for 20 hr, and then harvested for Tip110 expression by Western blotting using anti-His antibody (Input) or immunoprecipitated using an anti-HA antibody, followed by Western blotting using anti-HA antibody after the SDS-PAGE was run for a long time to capture polyubiquitinated smear above the Tip110 band. (E) 293T were transfected with UB.HA and Tip110.His, cultured for 24 hr, treated with TNF-α (10 ng/ml) for 30 min, and then harvested for expression of total ubiquitin, Tip110, and β-actin by Western blotting using anti-HA and anti-His antibody (Input) or immunoprecipitated using anti-His antibody followed by the same Western blotting to identify the amount of the ubiquitinated Tip110. An Alexa Fluor 488 (green), 555 (red), and 633 (purple) secondary antibodies were used to detect the Tip110, UB, and β-actin, respectively. Tip110 bands in the input appeared in yellow due to the overlay of green (Tip110) and red (ubiquitin) fluorescence. pcDNA3 was used to equalize the total amount of DNA among the transfection, and pGFP expression vector was added to ensure comparable transfection efficiencies among all transfections. β-actin was used as an internal loading control. The data were means ± SE from triplet samples and representative of three independent experiments. HC, IgG heavy; LC, IgG light chain. ***P < 0.001.