FIGURE 6.

RARγ phosphorylation and the effect of p38 MAPK inhibitor on RARγ subcellular localization. A, transfected RARγ is phosphorylated. HEK293T cells cultured in 6-well plates were transfected with RARγ or RARγ(S77/79A) expression vector (1 μg/well) in the presence or absence of SB203580 (10 μm). Lysates prepared from RARγ-transfected cells were also treated with alkaline phosphatase (AP). Cell lysates were subject to Western blot analysis using anti-RARγ antibody. B, regulation of RARγ phosphorylation by RXRα and RA. HEK293T cells cultured in 6-well plates were transfected with RARγ or RARγ (S77/79A) expression vector (0.5 μg/well) with or without RXRα (1.5 μg/well) and treated with RA for 1 h. Cell lysates were subject to Western blot analysis using anti-RARγ antibody. C and D, inhibition of RARγ cytoplasmic accumulation by SB203580. HeLa cells cultured in 24-well plates were transfected with Myc-RARγ (C) or Myc-RARγ/AB (D) (100 ng/well), and then treated with or without SB203580 (10 μm) for 1 h. Cells were stained with anti-Myc antibody and examined by fluorescence microscopy. About 80% of transfected cells showed RARγ or RARγ/AB nuclear staining when treated with SB203580. E, mutation of phosphorylation sites in RARγ/AB impairs its cytoplasmic localization. HeLa cells cultured in 24-well plates were transfected with Myc-RARγ/AB(S77/79A) (100 ng/well). Cells were stained with anti-Myc antibody and examined by fluorescence microscopy. F, SB203580 inhibits serum-induced RARγ cytoplasmic localization. H460 cells cultured in serum-free medium for 24 h were stimulated with serum (10%) in the presence of SB203580 (10 μm) for 6 h. Cells were immunostained using anti-RARγ antibody.