FIG. 2.
TVBS1 is produced as two distinct receptor types. (A) Aliquots of [35S]cysteine-labeled protein lysate prepared from transfected human 293 cells that express TVBS1(ΔDD) were subjected to three rounds of sequential immunoprecipitation with protein A-Sepharose beads loaded with either SUB-rIgG (lanes 1 to 3) or SUE-rIgG (lanes 5 to 7) and then with SUE-rIgG (lane 4) or SUB-rIgG (lane 8). The precipitated TVB proteins were visualized by autoradiography. (B) Flow cytometric analysis of 293 cells expressing TVBS1 (ΔDD) was performed with SUB-rIgG or SUE-rIgG, followed by a fluorescein isothiocyanate-conjugated secondary antibody. The results of a representative experiment performed in triplicate are shown as the mean fluorescence values of each cell population with the standard deviations of the data indicated with error bars. (C) An experiment similar to that described for panel A was performed with extracellular supernatant containing sTVBS1. In this case, the immunoprecipitated proteins were detected by immunoblotting with SUB-rIgG and an HRP-conjugated secondary antibody followed by enhanced chemiluminescence.