Tsg signals via the TGF-β–specific Smad2/3 in CD4⁺ T cells. (A) Primary CD4⁺ T cells or primed alloreactive CD4⁺ T-cell lines were cultured with BMP4 (100 ng/mL), Tsg (2 μg/mL), or control for 1 hour. Cell lysates were prepared and equal amounts (250 μg/sample) were used for immunoprecipitation with Smad1-specific antibody, followed by SDS-PAGE and Western blot with mAbs specific for p-Smad1 and Smad1. Results are representative of 4 independent experiments. DHL6 cell line was used as a positive control for Smad1 phosphorylation. (B) Primary CD4⁺ T cells or primed alloreactive CD4⁺ T-cell lines were cultured as in panel A and cell lysates (75 μg/sample) were analyzed by SDS-PAGE and Western blot with mAbs specific for p-Smad2 and Smad2. (C) Primed alloreactive CD4⁺ T-cell line was cultured for 1 hour with either media, TGF-β (2 ng/mL), Tsg (2 μg/mL), Tsg (5 μg/mL), or the combination of TGF-β (2 ng/mL) plus Tsg (2 μg/mL). Nuclear extracts were prepared and MSAs were done using Smad3/4 consensus oligonucleotides (Santa Cruz Biotechnology). Results are representative of 2 independent experiments.