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Fig. 4 | Cancer Cell International

Fig. 4

From: Circadian protein BMAL1 promotes breast cancer cell invasion and metastasis by up-regulating matrix metalloproteinase9 expression

Fig. 4

The interaction between BMAL1 and p65. a, b HEK 293T cells transfected with Flag-p65 only, HA-BMAL1 only or with Flag-p65 and HA-BMAL1 were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot with anti-HA antibody or vice versa. c HEK 293T cells transfected with HA-BMAL1 only, Flag-p50 only or with HA-BMAL1 and Flag-p50 were subjected to immunoprecipitation with anti-HA antibody followed by Western blot with anti-Flag antibody or vice versa. d ZR-75-30 cells were subjected to immunoprecipitation with anti-BMAL1 antibody followed by Western blot with anti-p65 antibodies. IgG was used as a negative control. e Direct interaction between BMAL1 and p65 as detected using a mammalian two-hybrid system. BMAL1 and p65 were expressed from pACT-BMAL1 and pBIND-p65, respectively, whereas the empty vectors pACT and p-BIND were expressed as controls, as indicated with the pG5-luc reporter in COS-7 cells. Cells were transfected with pBIND-ID and pACT-MyoD as a positive control. Luciferase activity was measured after 24 h of transfection. **p < 0.01 compared with cells transfected with pACT and pBIND. f Localization of Bmal1 and p65 in MCF-7. MCF-7 cells transfected with GFP-p65 and HA-BMAL1 were stained with rabbit anti-HA antibody and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit IgG. GFP-p65 appeared as a green signal when visualized by fluorescence microscopy. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). All experiments were repeated at least three times. Data are presented as means ± SD (p < 0.05, significant; *, p < 0.05; **, p < 0.01)

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