Fig. 4

The interaction between p65 and TIMELESS. a 293T cells were transfected with Flag-TIMELESS alone with or without GFP-p65 for 24 h, and then subjected to the immunoprecipitation with anti-GFP-antibody followed by western blot with anti-Flag antibody. b 293T cells were transfected with the indicated vectors for 24 h and then the immunoprecipitation was performed. c Extract from T47D cells was immunoprecipitated with anti-IgG or anti-p65 antibodies followed western blot with anti-TIMELESS antibody. d T47D cells were transfected with GFP-p65 together with or without Flag-TIMELESS for 24 h. TIMELESS was stained with anti-Flag antibody and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit IgG, and p65 was visualized with green fluorescence. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). e The direct interaction between p65 and TIMELESS was identified through mammalian two-hybrid systems. ZR-75–30 cells were transfected with the indicated vectors for 36 h and the activity of pSG5 luciferase was measured. Each bar represents the mean ± s.d. from three independent experiments. “*”, p < 0.05; “**”, p < 0.01