IGF-1R protein differentially accumulates in the nuclei of TAO orbital fibroblasts and derives from the fibroblast surface.

(A) 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 “Methods” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. (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. (C) 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. (D) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. (E) Control and GD fibroblasts were subjected to ¹²⁵I-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 “Methods” and subjected to quantification of radioactivity. Results are representative of three experiments performed.