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Fig. 4. Characteristics of the INS-1 stable cell line expressing DN-SREBP-1c. (A) Phase-contrast images (top) and immunofluorescence staining (bottom) with a rabbit polyclonal antibody against SREBP-1 showed that DN-SREBP-1c protein was induced by doxycycline in an all-or-none manner. DN-SREBP-1c*23 cells were cultured with (+Dox) or without (-Dox) 500 ng/ml doxycycline for 48 hours. (B) Gel-shift assay of nuclear extracts from DN-SREBP-1c*23 cells using the SRE sequence of the IRS2 promoter. DN-SREBP-1c*23 cells were cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours, in the presence (+Dox) or absence (-Dox) of 500 ng/ml doxycycline. Monoclonal SREBP-1-specific antibody was used for the supershift experiment. Experiments were repeated two to three times with similar results. (C) EMSA of nuclear extracts from non-induced DN-SREBP-1c*23 cells using the SRE sequence of the IRS2 promoter. DN-SREBP-1c*23 cells were cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours. Monoclonal SREBP-2-specific antibody was used for the supershift experiment. Experiments were repeated twice with similar results.





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