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Fig. 1. RXR
shuttles between the nucleus and the cytoplasm. (A) NIH3T3 cells were transfected with GFP-RXR expression vector and HeLa cells were then seeded onto the same coverslip. Before being fused by PEG to form heterokaryons, cells were cultured in medium containing cycloheximide (CHX) to inhibit new protein synthesis. Fused cells were treated with or without 9-cis retinoic acid (9-cis-RA, 1 µM) for 1 hour, then immunostained with Hoechst 33258 and tubulin antibody followed by Cy3-conjugated secondary antibody to display the nuclei and the whole cells simultaneously. The murine NIH3T3 nuclei gave a characteristic staining of intranuclear bodies (speckles) and the human HeLa nuclei displayed a diffuse pattern indicated by the arrows. (B) Subcellular localization of RXR in response to 9-cis retinoic acid. MGC80-3 cells were treated with or without 9-cis retinoic acid for the indicated times, fixed and immunostained with anti-RXR antibody followed by FITC-conjugated secondary antibody. Cells were also stained with propidium iodide (PI, Sigma, 50 µg/ml) to visualize nuclei. Images were visualized using a confocal microscope and the two images were merged (Overlay). (C) 9-cis retinoic acid-induced redistribution of RXR shown by western blotting in MGC80-3 cells. Cells were treated with 9-cis retinoic acid for the times indicated with nuclear and cytoplasmic fractions prepared as described in Materials and Methods. Different protein portions were then subjected to western blotting with anti-RXR antibody. Lamin B1 and -tubulin were used to quantify the amount of protein loaded.
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