Glucocorticoid treatment of thymocytes blocks NF-κB DNA binding, up-regulates GILZ expression, and induces its association with p65.

(A) EMSA analysis of NF-κB binding factors from untreated thymocytes (lane 1), or thymocytes cultured for 4 hours with 100 nM DEX (lane 2), or anti-CD3 mAb 1 μg/mL (lane 3), or the combination of anti-CD3 and DEX (lane 4). (B) Western blot analysis of GILZ. Lane 1: untreated thymocytes; lane 2: thymocytes treated with 100 nM DEX; lane 3: thymocytes treated with 1 μg/mL anti-CD3 mAb; lane 4: thymocytes treated with DEX and anti-CD3 mAb. (C,D) Immunoprecipitation of p65 and coimmunoprecipitation of GILZ. Mouse thymocytes were cultured in the presence or absence of DEX; cellular protein was collected 4 hours after stimulation and immunoprecipitated with anti-p65 antiserum (3 μL). Immunoblotting with anti-p65 antiserum (C) or anti-GILZ (D) was performed as described in the text. Lane 1: immunoprecipitation of untreated thymocytes; lane 2: immunoprecipitation of DEX-treated thymocytes; lane 3: total lysate from untreated thymocytes; lane 4: total lysates from DEX-treated thymocytes.