Figure 1

Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204. The transformants were grown on an SD plate lacking adenosine and uracil for 48 h. Murine p53 and SV40 large T antigens were used as a positive control. Two panels show yeast colonies showing the interaction of Tat and NUCKS1. (B) Ectopically expressed Tat interacts with ectopically expressed NUCKS1 in HEK293 cells. Two micrograms of the Flag-Tat, Flag-Vpr and Flag-Nef expression vector were cotransfected with the V5-NUCKS1 expression vector into HEK293 cells cultured in 100 mm plates. Forty-eight hours after transfection, cell lysates were immunoprecipitated with an anti-Flag monoclonal antibody (M2). Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-V5 monoclonal antibody. The Flag- or V5-tagged pcDNA3 plasmid was added to equalize the total amounts of DNA. (C) Coimmunoprecipitation assay between endogenous NUCKS1 and ectopically expressed Tat protein. The lysate from Tat-expressing HEK293T cells was immunoprecipitated with anti-Flag and -Pol II antibodies, and the interaction was assessed with Western blotting using anti-NUCKS1 or anti-Flag antibody.