Figure 1

Tax interacts with SUV39H1 in vitro. (a) HEK293T cells were transiently cotransfected with GST-SUV39H1 or GST and Tax. After 48 h, the cells were lysed and the proteins were affinity purified with Glutathione Sepharose 4B. Purified proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-Tax antibody Lt-4 (top panel). Expression of transduced proteins was confirmed by immunoblot analyses of whole cell lysates using respective antibodies (lower panels). (b) HEK293T cells were transiently co-transfected with expression plasmids, GST-SUV39H1 or GST and Tax. After 48 h, the cells were lysed and the proteins were immunoprecipitated with Lt-4. The immunoprecipitates were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-SUV39H1 or anti-GST antibody (upper panels). Expression of proteins was confirmed by immunoblot analyses of whole cell lysates using respective antibodies (lower panels). (c) Direct interaction between SUV39H1 and Tax. Bacterially expressed GST-SUV39H1 and GST were purified with Glutathione Sepharose 4B, and histidine-tagged wild type Tax (His-Tax) was purified with ProBond Resin (Promega). GST-SUV39H1 and GST were bound to Glutathione Sepharose 4B, and mixed with purified His-Tax in PBS. After centrifugation, proteins bound to Glutathione Sepharose 4B were separated by electrophoresis, transferred to a PVDF membrane, and probed with anti-Tax antibody. As a control, an aliquot of purified His-Tax was run in lane 4. IP, immunoprecipitation; IB, immunoblot; H.C., heavy chain