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. 2009 Feb 25;83(9):4652–4669. doi: 10.1128/JVI.02408-08

FIG. 4.

FIG. 4.

EBNA3C colocalizes with Mdm2. (A) Human embryonic kidney cell line HEK 293T (top panel) or human breast epithelial cell line U2OS (middle panel) plated on coverslips and transfected with pRK5-Flag-Mdm2, encoding Flag-tagged Mdm2, and pCDNA-EBNA3C, encoding untagged full-length EBNA3C, using Lipofectamine 2000. Cells of an EBV-transformed LCL, LCL1, expressing EBNA3C, were air dried onto slides. (B) U2OS cells were transfected with GFP-tagged EBNA3C fragments, residues 1 to 365 (top panel) and 366 to 620 (bottom panel), with Flag-tagged Mdm2. Cells were fixed using a 1:1 mixture of acetone and methanol. Ectopically and endogenously expressed Mdm2 was detected using M2 antibody (1:100 dilution) and SMP14 (1:100 dilution), respectively, followed by anti-mouse Alexa Fluor 594 (red), and full-length EBNA3C was detected using EBNA3C-reactive rabbit serum (1:150 dilution) followed by anti-rabbit Alexa Fluor 488 (green). The nuclei were counterstained using DAPI. The images were sequentially captured using an Olympus confocal microscope. All panels are representative pictures from similar repeated experiments.