Transfected 293T cells or reactivated iSLK/rKSHV.219 cells [sodium butyrate (1 mM) and doxycycline (1 μg/ml)] were harvested and lysed with NP-40 buffer [50 mM tris-HCl (pH8.0), 150 mM NaCl, and 1% NP-40] supplemented with protease inhibitor cocktail (Roche). WCLs were sonicated, centrifuged, and precleared with protein A/G agarose for 1 hour. Precleared cell lysates were then incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich) for 4 hours at 4°C or anti–importin β1/anti-kRTA antibodies overnight following protein A/G agarose incubation for 1 hour at 4°C. The agarose beads were washed extensively, and protein was eluted with 1× SDS-loading buffer by boiling for 10 min. Samples were then subjected to SDS-PAGE and immunoblotting.

For GST pulldown with importin β, 293T cells were transfected with plasmids containing RTA-WT or RTA-DD and then lysed in binding buffer [20 mM tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, and 0.5% NP-40] plus protease inhibitor cocktail (Roche). Cell lysates were centrifuged and then precleared with glutathione beads. Precleared cell lysates were incubated with glutathione resin loaded with GST or GST–importin β (~4 μg of protein). After incubation at 4°C for 4 to 6 hours, the resin was washed three times in binding buffer and then proteins were eluted, resolved by SDS-PAGE, and analyzed by immunoblotting. All immunoblotting analyses were performed using the indicated primary antibodies (1:1000 dilution) and IRDye800-conjugated secondary antibodies (1:10,000 dilution) (LI-COR). Proteins were visualized using an Odyssey infrared imaging system (LI-COR).

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