Fig. 5. RXRa serves as a carrier for TR3 translocation initiated by 9-cis retinoic acid. (A) Detection of TR3 translocation mediated by RXR in living MGC80-3 cells. Living cells transfected with GFP-TR3 alone or together with RXR were treated with 9-cis retinoic acid at different time points as indicated. The GFP-TR3 translocation from the nucleus to the cytoplasm was visualized under a fluorescent microscope. To detect the effect of LMB on TR3 translocation, transfected cells were pre-treated with LMB for 2 hours, followed by treatment of 9-cis retinoic acid for the indicated times. (B) Repression of endogenous RXR and TR3 by antisense RXR, antisense TR3 or TR3-siRNA. Cells were stably transfected with different expression vectors or transiently transfected with siRNA as described in Materials and Methods. Expression level of endogenous RXR or TR3 was detected by western blotting. Empty vector and scrambled-siRNA were used as controls. tubulin was used to quantify the amount of protein loaded in each lane. (C) Effects of antisense RXR, antisense TR3 and TR3-siRNA on the translocation of RXR and TR3. MGC80-3 cells were transfected with different expression vectors or siRNAs as described in B and then treated with 9-cis retinoic acid (1 µM) for 6 hours. Nuclear (N) and cytoplasmic (C) fractions were subjected to western blotting, probed with anti-TR3 or anti-RXR antibody as indicated. tubulin and lamin B1 were used as loading controls. Scrambled-siRNA was used as positive control.