A Unique SUMO-2-Interacting Motif within LANA Is Essential for KSHV Latency
Figure 2
The LANASIM deletion reduces the co-localization of LANA with SUMO-2 on chromatin DNA.
HEK293 cells co-transfected GFP-SUMO-2 with vector (A) alone, wild type (WT) or SIM-deleted (ΔSIM) mutant of LANA NC with myc tag (B) was individually cultured on coverslips, fixed with 3% paraformaldehyde, and then stained with anti-myc antibody as indicated. Bottom panels show the magnified view and arrowheads point examples of LANA and SUMO-2 co-localization on chromosome DNA within the outline region. Nuclei were counterstained with DAPI. Scale bars, 2 µm. Right panel shows schematic of co-localization (yield white) of LANA (red) and SUMO-2 (green) with host chromatin DNA (blue) and the percentage of co-localization from 50 counted cells. (C) Sequential ChIP was performed first with anti-GFP antibody or mouse IgG followed by a second ChIP with anti-myc antibody. Quantitative PCR was performed using TR specific primer. (D) Hypoxia reduces the co-localization of LANA with SUMO-2. BC3 cells were subjected to hypoxia (1% oxygen) treatment for overnight. Endogenous LANA and SUMO-2 were individually stained by LNA1 (rat) and SUMO-2 (rabbit) antibodies. SUMO-2 non-colocalization with LANA was highlighted by dot box. Percentage of co-localization from 10 counted cells.