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. 1999 Feb 2;96(3):1042–1047. doi: 10.1073/pnas.96.3.1042

Figure 5.

Figure 5

Ad E3 14.7K affects the intracellular localization of FIP-3. 293 cells were transfected with plasmids expressing various combinations of pcDNA-T7-FIP-3 and pcDNA-FLAG-14.7K. The nuclear and cytoplasmic fractions were prepared by Dounce homogenization in hypotonic buffer, and each was analyzed for FIP-3 protein 24 hours later by Western blots using antibody to the T7 tag conjugated with horseradish peroxidase (Novagen) at a dilution of 1:2,000 for 1.5 hours at room temperature. The blots were washed 3–4 times in PBS with 0.1% Tween 20 and were developed with enhanced chemiluminescence reagents (Boehringer Mannheim). Lanes: 1 and 2, negative controls that contain 5 μg of pcDNA-T7 and 3 μg of pcDNA-14.7K, respectively; 3–7, 1 μg of pcDNA-FIP-3; 4–7, increasing amounts of pcDNA-14.7K; 4, 1 μg; 5, 2 μg; 6, 3 μg; 7, 4 μg. All transfections were normalized to contain 5 μg of DNA by the addition of pcDNA-T7.