Tbx1 Regulates the BMP-Smad1 Pathway in a Transcription Independent Manner
Figure 2
a) Western blot analysis of affinity-purified nuclear extracts from P19-Tbx1-PA (lane 1) and P19-PA (lane 3) cells, compared non-purified nuclear extracts of P19-Tbx1-PA cells (lane 2), using an antibody anti-Phospho-SMAD1/5/8. b) Western blot analyses of reciprocal coimmunoprecipitation (IP) experiments using the antibodies indicated. NIH3T3 cells were co-transfected transiently with Tbx1-cmyc and Smad1-Flag expression vectors. c) Western blot analyses of nuclear extracts from wild type E9.5 mouse embryos immunoprecipitated with an anti-Tbx1 antibody (lanes 1 and 2) or with anti-rabbit IgG (lane 3) and revealed with an anti-SMAD1 antibody (lane 1 and 3) and an anti-Phospho-SMAD1/5/8 antibody (lane 2). d) Immunoblotting with anti-Smad1 antibody of nuclear extracts of wild type mouse embryos (E9.5) coimmunoprecipitated with anti-Tbx1 (NE-IP), total nuclear extracts (NE), total cytoplasmic extracts (CE), and cytoplasmic extracts coimminoprecipitated with anti-Tbx1 (CE-IP). e) Coimmunoprecipitation of WT TBX1, TBX1G145R, TBX1F148Y, TBX1G310S and Flag-SMAD1 transiently transfected in NIH 3T3 cells. The TBX1G145R and TBX1F148Y mutants physically interact with Smad1, while TBX1G310S is unable to bind Smad1 indicating that Glycine 310 is important for this interaction. f) Luciferase assay using Cos7 cells transfected with the SMAD-responsive reporter NTK-tetramer-luc. Transfected SMAD1 activates the reporter while increasing amounts (5 to 100 ng) of co-transfected TBX1 suppresses it.