Nestin and Steroid Receptor Figure 1. Nestin and GR colocalize in embryonic tissue of mice. (A) Horizontal section through the head of a mouse embryo (E 14.5); paraffin section stained with hematoxilin-eosin; (B–E) Immunoperoxidase labelling of GR and Nes on consecutive sections demonstrates expression in glia (B and C) and muscle fibres at vibrissae (D and E). (F–G) Fluorescence double labelling demonstrates co-localization of GR and Nes in glia (F1–F3) and in developing muscles adjacent to a hair follicle (G1–G3). Bars in A–E = 100 mm; G1 = 20 mm; G3 = 10 mm. doi:10.1371/journal.pone.0006084.g001 Subsequently, we asked whether anchoring of GR to the IF system could be modulated in dependence of nestin. Western blots of total cell lysates revealed a strong nestin signal in the anchoring competent C6D8 and C6K23 cells (Figure 5A, lanes 1 and 3), whereas only a weak signal was present in total lysates of the IF deficient C6D10 cells (Figure 5A, lane 2). Immunoprecipitation experiments done with the detergent soluble fraction of C6D10 cells showed no co-precipitation of GR and nestin, despite the fact that significant amounts of nestin were precipitated (Figure 5B, lanes 1 and 2). In contrast, only low amounts of nestin were immunoprecipitated from lysates of C6D8 cells, and these complexes contained GR (Figure 5B, lane 4). The data indicate that nestin in the IF deficient C6D10 cells is soluble and not associated with GR. In contrast, nestin in the vimentin positive PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e6084