Skip to main content
. 2009 Mar 4;83(10):4871–4883. doi: 10.1128/JVI.02286-08

FIG. 1.

FIG. 1.

(a) Levels of Necdin are much lower in EBV- and KSHV-positive B cells than in control cells when compared at the mRNA level by quantitative real-time PCR. The specific primers for Necdin used were as follows: sense, 5′-GAGTTTGCCCTGGTCAAAGC-3′; antisense, 5′-CATGGGCATACGGTTGTTGAG-3′. PCR amplification yielded a 92-bp product. For β-actin, the primers were as follows: sense, 5′-GCTCGTCGTCGACAACGGCTC-3′; antisense, 5′-CAAACATGATCTGGGTCATCTTCTC-3′. A melting curve analysis was performed to verify the specificity of the products, and the values for the relative quantitation were calculated by the ΔΔCT method. (b) EBNA3C affects the localization of Necdin. Twenty million transfected cells were harvested and washed twice with ice-cold PBS, followed by resuspension of the cell pellet in hypotonic buffer A (10 mM HEPES-K+ [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 0.5 dithiothreitol) in the presence of PIC. Cells were pelleted by spinning them at 1,000 rpm for 5 min. The cells were lysed in ice-cold 0.5% NP-40-containing buffer A with PIC on ice for 10 min. The nuclei were pelleted by centrifugation at 3,000 rpm for 2 min at 4°C. The supernatant (cytoplasmic protein) was harvested and frozen at −80°C for use. The nuclear pellets were washed with buffer A (without NP-40), followed by resuspension in buffer C (20 mM HEPES-K+ [pH 7.9], 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 dithiothreitol, 25% glycerol) with PIC. Nuclei were incubated on ice for 30 min and vortex mixed periodically. Supernatants containing nuclear protein were collected by spinning at 14,500 rpm for 10 min at 4°C and then snap frozen for further use. In the presence of EBNA3C, Necdin localization changes from primarily cytoplasmic to primarily noncytoplasmic. Soluble nuclear (N) and cytoplasmic (C) fractions were compared for cells coexpressing Necdin with EBNA3C (lanes 3 and 4) and cells expressing Necdin alone (lanes 5 and 6). Lanes 1 and 2 represent 293T cells as negative controls. (c) Expression levels of EBNA3C. Antibodies to nuclear protein Sp1 (1C6; Santa Cruz, Inc., Santa Cruz, CA) and cytoplasmic protein Hsp70 (BD Transduction Laboratory) were used as markers. (d and e) The localizations of HSP70, which is primarily cytoplasmic, and Sp1, which is nuclear, show the efficiency of cellular fractionation in nuclear and cytoplasmic fractions. Error bars indicate standard deviations.