Analysis of JK cell TβR-I receptors. (A) Immunocomplexes of JK cell TβR-I fail to phosphorylate recombinant GST-Smad3. Mv1Lu (Mv1), human HT1080 fibrosarcoma (HT), human MDA-MB-231 breast cancer (231), or JK cells were incubated in the absence or presence of TGF-β1 for 10 minutes at 37°C. Triton X-100 solubilized cell extracts were prepared and subjected to immunoprecipitation with anti-TGF–β type II receptor antibodies, and the resulting immunocomplexes were incubated in the presence of recombinant GST-Smad3 and [γ-³²P]ATP as described in Materials and Methods. Data shown is a representative autoradiograph depicting TGF-β–stimulated phosphorylation of recombinant GST-Smad3. (B) Loss of cell surface expression of TβR-I receptors in JK cells. Mv1Lu or JK cells (5 × 10⁶ cells/tube) were incubated in the presence of 250 pmol/L [¹²⁵I]TGF-β1 for 90 minutes at 4°C before addition of disuccinimidyl suberate to cross-link the cytokine/receptor complexes. Triton X-100 solubilized cell extracts were prepared and subjected to immunoprecipitation with anti-TβR–I or -TβR–II receptor antibodies as described in Materials and Methods. Data shown is a representative autoradiograph depicting iodinated TGF-β1 bound to its TβR-I and TβR-II receptors as indicated. (C) TβR-I mRNA is expressed normally in JK cells. A total of 5 μg of total mRNA was hybridized to a³²P-radiolabeled probe corresponding to nucleotides 226-1536 of the TβR-I cDNA. Data shown is a representative autoradiograph of the ∼6.5 kb TβR-I mRNA transcript in Mv1Lu and JK cells.