Figure 3.
Figure 3. Detection by flow cytometry of IGF-2 receptor expression in fetal liver cells. (A) Production and secretion of IGF-2–hFc in transfected 293T cells. The upper panel shows a schematic of the plasmid expressing the human prepro–IGF-2 protein fused to a human IgG1 Fc fragment. The bottom panels show Western blots of the 48-hour conditioned medium of 293T cells transfected either by control or IGF-2–hFc–expressing vectors probed with antibodies against human IGF-2 (left) or human IgG1 (right). (B) Most fetal liver Lin-Sca-1+ cells specifically bind IGF-2–hFc. Total day 15 fetal liver cells were incubated at 4°C for 30 minutes with conditioned medium from control transfected 293T cells (iii) or cells transfected with the IGF-2–hFc expression vector (iv, v); the latter medium contained about 1 μg/mL IGF-2–hFc. Cells were then stained with anti-human IgG-PE, followed by the biotinylated Lin+ antibody cocktail, streptavidin-APC, anti-Sca-1–FITC, and propidium iodide (PI). For FACS analysis of total fetal liver, single cells that excluded PI were further gated by forward scatter/side scatter (i) to include only nucleated cells. Lin- cells were gated as the lowest 5% of APC-stained cells (ii). The gated cells were then analyzed for the binding of IGF-2–hFc and the level of Sca-1 (iii-v). (C) Western blot of total fetal liver cells sorted based on binding of IGF-2–hFc. (i, ii) The purity of IGF-2–hFc+ and IGF-2–hFc- fetal liver cells sorted after staining with IGF-2–hFc and anti-hIgG1–PE; panel i is the reanalysis of the sorted IGF-2–hFc- cells and panel ii, the reanalysis of the sorted IGF-2–hFc+ cells. In panel iii, antibodies against IGF1R (Cell Signaling Technology, Beverly, MA) and IGF2R (a gift from Dr Stuart Kornfeld) were used to detect the expression of these IGF-2 receptors in IGF-2–hFc+ and IGF-2–hFc- fetal liver cells. These results confirmed the specificity of IGF-2–hFc binding because cells unable to bind the IGF-2–hFc fusion protein did not express these 2 principal IGF-2 receptors. Blotting by anti–β-tubulin (Sigma) served as the loading control. (D) RT-PCR analysis of cDNA isolated from total fetal liver cells sorted based on binding of IGF-2–hFc. RT-PCR was used to detect the mRNA levels of IGF1R, IGF2R, and insulin receptor in IGF-2–hFc+ and IGF-2–hFc- fetal liver cells sorted as in panel C. Expression of GAPDH was measured as an indicator of the amount of RNA.

Detection by flow cytometry of IGF-2 receptor expression in fetal liver cells. (A) Production and secretion of IGF-2–hFc in transfected 293T cells. The upper panel shows a schematic of the plasmid expressing the human prepro–IGF-2 protein fused to a human IgG1 Fc fragment. The bottom panels show Western blots of the 48-hour conditioned medium of 293T cells transfected either by control or IGF-2–hFc–expressing vectors probed with antibodies against human IGF-2 (left) or human IgG1 (right). (B) Most fetal liver Lin-Sca-1+ cells specifically bind IGF-2–hFc. Total day 15 fetal liver cells were incubated at 4°C for 30 minutes with conditioned medium from control transfected 293T cells (iii) or cells transfected with the IGF-2–hFc expression vector (iv, v); the latter medium contained about 1 μg/mL IGF-2–hFc. Cells were then stained with anti-human IgG-PE, followed by the biotinylated Lin+ antibody cocktail, streptavidin-APC, anti-Sca-1–FITC, and propidium iodide (PI). For FACS analysis of total fetal liver, single cells that excluded PI were further gated by forward scatter/side scatter (i) to include only nucleated cells. Lin- cells were gated as the lowest 5% of APC-stained cells (ii). The gated cells were then analyzed for the binding of IGF-2–hFc and the level of Sca-1 (iii-v). (C) Western blot of total fetal liver cells sorted based on binding of IGF-2–hFc. (i, ii) The purity of IGF-2–hFc+ and IGF-2–hFc- fetal liver cells sorted after staining with IGF-2–hFc and anti-hIgG1–PE; panel i is the reanalysis of the sorted IGF-2–hFc- cells and panel ii, the reanalysis of the sorted IGF-2–hFc+ cells. In panel iii, antibodies against IGF1R (Cell Signaling Technology, Beverly, MA) and IGF2R (a gift from Dr Stuart Kornfeld) were used to detect the expression of these IGF-2 receptors in IGF-2–hFc+ and IGF-2–hFc- fetal liver cells. These results confirmed the specificity of IGF-2–hFc binding because cells unable to bind the IGF-2–hFc fusion protein did not express these 2 principal IGF-2 receptors. Blotting by anti–β-tubulin (Sigma) served as the loading control. (D) RT-PCR analysis of cDNA isolated from total fetal liver cells sorted based on binding of IGF-2–hFc. RT-PCR was used to detect the mRNA levels of IGF1R, IGF2R, and insulin receptor in IGF-2–hFc+ and IGF-2–hFc- fetal liver cells sorted as in panel C. Expression of GAPDH was measured as an indicator of the amount of RNA.

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