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. 2012 Feb 13;122(3):873–885. doi: 10.1172/JCI61498

Figure 3. Altered ligand/receptor assembly and upregulated TAK1 phosphorylation in the absence of Tgfbr2.

Figure 3

(A) Immunoblotting analysis of immunoprecipitation products derived from IgG control beads with Tgfbr2fl/fl or Tgfbr2fl/fl;Wnt1-Cre MEPM cell extracts (lanes 1 and 2, respectively), Tgfbr2fl/fl MEPM cell extracts (lane 3), and Tgfbr2fl/fl;Wnt1-Cre MEPM cell extracts (lane 4) using the indicated antibodies. The bar graphs show the ratios of TβRIII and β-spectrin after quantitative densitometry analysis of immunoblotting data. Three samples were analyzed for each experiment. Error bars represent SD. (B) Immunoblotting analysis of immunoprecipitation products using anti–His-tag antibody derived from IgG control beads or MEPM cell extracts from Tgfbr2fl/fl or Tgfbr2fl/fl;Wnt1-Cre mice after overexpression of His-tagged Tgfbr1 and FRP-tagged Tgfbr3, with (+) or without (–) TGF-β2 treatment. (C) Cross-linking analysis after treatment with radioactive iodine-125 (125I) TGF-β2 in primary MEPM cells from Tgfbr2fl/fl and Tgfbr2fl/fl;Wnt1-Cre mice. (D) Immunoprecipitation products derived from IgG control beads (lane 1), Tgfbr2fl/fl MEPM cell extracts (lane 2), and Tgfbr2fl/fl;Wnt1-Cre MEPM cell extracts (lane 3) after cross-linking with 125I–TGF-β2. The top arrowhead indicates TβRII::TGF-β2 complex after IP with anti-TβRII antibody, and the bottom arrowhead indicates TβRI::TGF-β2 complex after IP with anti-TβRI antibody. The bracket indicates TβRIII::TβRI::TGF-β2 complex. (E) Immunoblotting analysis of immunoprecipitation products derived from IgG control beads (lane 1), Tgfbr2fl/fl MEPM cell extracts (lane 2), and Tgfbr2fl/fl;Wnt1-Cre MEPM cell extracts (lane 3) after cross-linking with 125I–TGF-β2. (F) Immunoblotting analysis of indicated molecules in primary MEPM cells from Tgfbr2fl/fl and Tgfbr2fl/fl;Wnt1-Cre mice cultured with TGF-β2 for indicated times. The bar graphs show the ratios of phosphorylated SMAD2 relative to SMAD2/3, phosphorylated TAK1 relative to TAK1, or phosphorylated p38 relative to p38 after quantitative densitometry analysis of immunoblotting data.