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. 2014 Jul 17;10(7):e1004274. doi: 10.1371/journal.ppat.1004274

Figure 8. LANA induces relocalization and SUMOylation of Sp100.

Figure 8

(A) EA.hy cells were transduced with an YFP labeled retroviral LANA expression construct (right panel) or a control retrovirus (left panel). YFP positive cells were enriched by FACS to reduce background of non-transduced cells. After subcellular fractionation, the localization of Sp100 in LANA was analyzed by western blotting. Successful fractionation was confirmed by detection of specific marker proteins (Hsp70, Sp1 and Vimentin). Abbreviations above each lane indicate the following fractions: cyto: cytoplasmic; memb: membrane-associated; nucpl: nucleoplasmic; chrom: chromatin-associated; matrix: insoluble matrix-associated. (B) Stable HIS-tagged SUMO-1 or SUMO-2 expressing and parental HeLa cells were transfected with pcDNA3-LANA or a control vector for 72 hours. Protein lysates were purified via Ni-NTA affinity chromatography and SUMOylated Sp100 or all precipitated SUMOylated proteins were detected by western blot analysis using Sp100 or HIS-specific antibodies (right panel). Input protein levels of Sp100 and HIS-tagged SUMO-1/-2 are shown in the left panel.