Figure 5.

FKBP51 SUMOylation is critical for its role as a GR cochaperone. (a) HEK293T cytoplasmic lysates expressing wt, K422R or 2KR FLAG-FKBP51 and HA-GR were used for in vitro ligand binding assay. Results are expressed as mean±S.E.M. (n=3). Lysates were analyzed by western blotting using the indicated antibodies. (b) HT22 cells transfected with YFP-GR plasmid with or without wt or K422R FKBP51 were stimulated with Dex 10 nM. Results are expressed as mean±S.E.M. of one representative experiment (n=3) (left panel). Representative images for each condition at t=0 s and t=3600 s are shown (right, upper panel). Cells transfected with YFP-GR plasmid with or without wt or K422R FKBP51 were analyzed by immunofluorescence using anti-FKBP51 antibody (right, bottom panel). (c) Lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG antibody and analyzed by western blotting using the indicated antibodies. (d) Lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-HA antibody and analyzed by western blotting using the indicated antibodies. (e) In vitro SUMOylated FKBP51 was incubated with FLAG-Hsp90 purified from HEK293T cells and immunoprecipitated with anti-FKBP51 antibody. Immunoprecipitates were analyzed by western blotting using the indicated antibodies (left panel). Pull-down assays were performed using in vitro SUMOylated FKBP51 and analyzed by western blotting using the indicated antibodies (right panel). (f) Lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-FKBP51 antibody (ab) or non-immune antibody (NI ab) and analyzed by western blotting using the indicated antibodies. *Non-specific. ^#IgG