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Fig. 4. Translocation of TR3 and RXR
in response to 9-cis retinoic acid. (A) Colocalization of endogenous RXR and TR3 in the mitochondria. MGC80-3 cells were treated with or without 9-cis retinoic acid (1 µM) for 12 hours and then immunostained with anti-TR3, anti-RXR and anti-Hsp60 antibodies followed with corresponding FITC-, Cy3-and Cy5-conjugated secondary antibodies to show endogenous TR3, RXR and Hsp60 proteins simultaneously. The fluorescent images were visualized with a confocal microscope, and the images were merged as indicated (Overlay). (B) MGC80-3 cells were treated with or without 9-cis retinoic acid (1 µM) for the indicated times. Cell extracts were prepared and immunoprecipitated with anti-RXR antibody, then subjected to SDS-PAGE, blotted, and probed with anti-TR3 antibody. The immunoprecipitated RXR used in each lane was quantified by western blotting with anti-RXR antibody. The same extract was applied to ascertain the position and expression of TR3 by western blotting with antibodies against TR3 (Input). IgG was used as negative control where no signal band was detected. (C) Distribution of TR3 and RXR in MGC80-3 cells. Nuclear, cytosolic and mitochondrial fractions were prepared as described in Materials and Methods, and the expression of RXR and TR3 in response to 9-cis retinoic acid at different time points was determined by western blotting. Mitochondrial Hsp60, nuclear protein Lamin B1 and cytosolic tubulin were detected as protein loading controls.
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