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Figure 3 | Journal of Biomedical Science

Figure 3

From: Suppressive Regulation of KSHV RTA with O-GlcNAcylation

Figure 3

Disruption of O -GlcNAcylation on T366 and T367 leads to an increase in the transactivation activity of K-RTA. (A) Nuclear localizations of the O-GlcNAc mutants were similar to the wild-type K-RTA, as shown by an immunofluorescence assay using M2-FLAG antibody. (B) Western blot analysis of protein extracts from HEK293 cells transiently transfected with expression plasmids for K-RTA or O-GlcNAc mutants. GAPDH served as a loading control. Similar protein expression levels and migration patterns were observed between wild-type K-RTA and the three O-GlcNAc mutants. (C) Luciferase reporter assay of the KSHV ORF57 promoter responding to K-RTA and O-GlcNAc mutants in HEK293 cells. Each firefly luciferase value was normalized to an internal control plasmid, pRL-TK. The normalized value for K-RTA was set to 100%. Data are presented as the mean ± SD from triplicate transfections. * denotes P < 0.05 (Student's t-test). Six independent experiments were performed and a representative result is shown. (D) Luciferase reporter assay of the KSHV PAN promoter responding to K-RTA and O-GlcNAc mutants in HEK293 cells. The procedure was carried out as described in (C), except that ORF57p was replaced with PANp. Six independent experiments were performed and a representative result is shown.

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