RA-enhanced expression and phosphorylation of Smad3. (A) Naive CD4⁺ T cells were activated with anti-CD3 alone or anti-CD3 and anti-CD28 for 12 h. (top) Total RNA was isolated, and expression of Smad3 mRNA was determined by quantitative real-time PCR. Data (means ± SEM) are representative of three independent experiments. (middle) Cell lysates were separated by SDS-PAGE gel, and total Smad3, p-Smad3, and p-Smad2 were analyzed by immunoblotting with specific antibodies. For quantification, the ratio of total Smad3/β-actin as well as the ratios of p-Smad3/β-actin and p-Smad2/β-actin were determined by densitometric analysis. Nuclear and cytoplasmatic extracts were analyzed by immunoblotting with specific antibodies. Cells were fixed and stained for confocal analysis by a Smad3 antibody. Data are representative of three to five independent experiments. (bottom) T reg cell conversion of naive CD4⁺ T cells from Smad3^−/− or Smad4^−/− or WT control mice activated with anti-CD3/CD28 in the presence of 0.3 ng/ml TGF-β or TGF-β plus 2.5 nM RA. (B, top) Representative FACS plots are shown (n = 3 independent experiments). (middle) Cell lysates of naive CD4⁺ cells from Smad3^−/− and WT control mice were cultured for 12 h (as in A, bottom) and separated by SDS-PAGE gel, and total Smad3 and Smad2 were detected by immunoblotting. Representative data out of four independent experiments are shown. (bottom) Naive CD4⁺ T cells from Smad4^−/− or WT control mice were fixed and stained by Smad3 antibodies and analyzed by confocal microscopy. The arrows indicate translocation of Smad3 into the nucleus. Representative data are shown (n = 3 independent experiments).