Nestin and Steroid Receptor Figure 5. GR from the cytoskeleton fraction of anchoring competent cells coimmunoprecipitates with nestin, vimentin, and HSC70. (A) SDS lysates of cells. Anchoring competent cells (C6D8 and C6K23) contain significant amounts of nestin. (B) Significant amounts of nestin are immunoprecipitated from the detergent soluble fraction of vimentin deficient C6D10 cells (B, lane 2) but not of vimentin positive C6D8 cells (B, lane 4); nestin solubilized from C6D8 cells coimmunoprecipitates with GR (B, lane 4). (C) Immunoprecipitation (IP) with the monoclonal vimentin antibody V9 shows co-precipitation of GR (C, lane 7); nuclei are excluded from the immunoprecipitate as indicated by the absence of lamin B; a monoclonal antibody to measles virus protein was used for control IP(C, lane 6); Figure 5C, upper part, shows the nitrocellulose sheet after protein transfer and staining with Amido black; the lower part shows the same membrane after incubation with goat anti-vimentin, rabbit anti-lamin B, and rabbit anti-GR antibodies and detection of bound antibodies with enzyme conjugated antibodies; (cyt) cytoskeleton fraction, (total) whole cell lysate; the relative position of the labelled proteins on the Amido black stained sheet is marked. (D) Western blot analysis (WB) indicates the presence of Nes in GR immunoprecipitates derived from the cytoskeleton fraction, but shows no coimmunoprecipitation of GR and tubulin (Tub). (E) GR derived from the cytoskeleton fraction coimmunoprecipitates with HSC70 (E, lane 2); GR precipitated from the supernatant lysis buffer coprecipitates also with HSP90 (E, lane 1). doi:10.1371/journal.pone.0006084.g005 Accordingly, only HSC70 was present in GR immunoprecipitates obtained from the cytoskeleton fraction (Figure 5E, lane 2). In contrast, GR which was immunoprecipitated from the detergent soluble fraction contained both, HSC70 and HSP90 (Figure 5E, lane 1). Association with HSC70 indicates that the filament bound receptor is inactive, but at an advanced stage in the activation cycle [40,41]. To further confirm this suggestion we assayed its reaction with certain antibodies that are specific for the nonactivated, respectively the activated form. GR is phosphorylated at three major sites on its N-terminus, namely Ser203, Ser211, and Ser226 in human GR, corresponding to Ser224, Ser232, and Ser246 in rat GR [42,43]. Phosphorylation of Ser211 is required for full transcriptional activity and correlates with a strong phospho-Ser211 staining in the nucleus, suggesting that Ser211 is a marker for activated GR in vivo [43,44]. In contrast, phosphorylation of Ser226 correlates with an increased receptor nuclear export in the absence of hormone. In addition, it decreases the transcriptional GR activity in response to a hormone, suggesting that it holds GR in the non-activated state [45,46]. Thus, we asked whether we could distinguish cytoskeleton bound and nuclear GR by reaction with these antibodies. A non-phospho specific GR antibody reacted with cytoplasmic as well as with nuclear GR in extracted C6D8 cells (Figure 10A). In contrast, the phospho-Ser226 antibody only detected cytoplasmic GR (Figure 10B), whereas the phospho-Ser211 antibody showed a dominant nuclear reaction in these cells (Figure 10C). In C6D8 cells treated with dexamethasone prior to Triton X-100 extraction, only a strong nuclear phospho-Ser211staining was present (Figure 10E), whereas phospho-Ser226 staining was lacking (Figure 10F). In accordance, GR could be immunoprecipitated with the phospho-Ser226 antibody only from lysates of C6D8 but not of C6D10 cells (Figure 10G). In contrast, the phospho-Ser211 antibody precipitated GR from lysates of both, CD10 and C6D8 PLoS ONE | www.plosone.org 7 June 2009 | Volume 4 | Issue 6 | e6084