<p>(<b>A</b>) Western blot analysis of nuclear and cytoplasmic IGF-1Rα in GD orbital fibroblasts before and following IGF-1 (10 nM) treatment for 16 h. Cells were subjected to subcellular fractionation as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. (<b>B</b>) Nuclear IGF-1Rα content in GD and control orbital fibroblasts before or following treatment with either IGF-1 (10 nM) or GD-IgG (15 µg/ml) for 16 hours. (<b>C</b>) Insulin fails to alter the nuclear content of IR or IGF-1Rα in GD orbital fibroblasts. Cells were treated with nothing or insulin (15 µg/ml) for 16 hrs. They were subjected to subcellular fractionation and Western blot analysis. (<b>D</b>) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. (<b>E</b>) Control and GD fibroblasts were subjected to <sup>125</sup>I-IGF-1 cross-linking with either the cell-impermeable agent, BS, or the cell permeable agent, DSS. They were then treated with IGF-1. Nuclei were separated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and subjected to quantification of radioactivity. Results are representative of three experiments performed.</p
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