FIG. 10.

HDAC6 is required for GR translocation and transcriptional activity. (A) Wild-type (WT) or HDAC6-deficient (knockout [KO]) MEFs or rescuants thereof were transiently cotransfected with an MTV-GRE-firefly luciferase reporter and a Renilla luciferase vector as a control. Dual luciferase activity was measured after treatment with dexamethasone, and the ratio of firefly luciferase activity to Renilla luciferase activity was used as relative luciferase activity. The rescuant cells express WT HDAC6, a catalytically inactive mutant HDAC6 (HD^m), or a non-ubiquitin-binding mutant of HDAC6 (Ub^m), as indicated. The data are the means of three independent experiments done in triplicate; P < 0.005 (WT versus KO). (B) The protein levels of GR and HDAC6 in the different cell lines were determined by immunoblotting with anti-GR and anti-HDAC6 antibodies. The cells were treated with dexamethasone (+) or not treated with dexamethasone (−). (C) Impaired GR translocation in the absence of HDAC6. Wild-type and HDAC6-deficient MEFs were cultured in hormone-free medium for 24 h and then stimulated with dexamethasone (+Dex) for 30 min. The localization of GR was determined by immunostaining with an anti-GR antibody.