Fig. 3.

Direct interaction between treacle and UBF. (A) HeLa cells were transfected with double-FLAG-tagged treacle (amino acids 717-1488) expression construct and analyzed by immunofluorescence staining using anti-FLAG and anti-Guα antibodies. Similar transfected cells were used for immunoprecipitation analysis by tumbling nuclear extract with anti-FLAG antibody-agarose. Protein samples were analyzed by Western blot enhanced chemiluminescence using anti-UBF antibody. The membrane was stripped and analyzed by using anti-Guα antibody. (B) Analysis of the treacle–UBF interaction by the yeast two-hybrid system in a double drop-out medium (-Trp, -Leu) that permits growth of cells transfected with pGBKT7 and pGADT7 constructs, and in a triple drop-out medium (-Trp, -Leu, -His) that screens for protein–protein interaction. The interaction of p53 and simian virus 40 large T antigen was used as a positive control.