GILZ does not affect p65 and P52 expression, I-κBα phosphorylation and degradation, or I-κB/NF-κB binding.

(A) Western blot analysis performed using protein lysates from untreated (lanes 1-4) or anti-CD3–treated (2 hours, lanes 5-8) 3DO cells (lanes 1, 5), empty vector–transfected clone PV6 (lanes 2, 6), GILZ-transfected clones ST7 (lanes 3, 7), and GIRL19 (lanes 4, 8). The nitrocellulose membrane was immunoblotted with anti-p65 antibody (i), or with anti-p52 antibody (ii), or with anti-β–tubuline mAb used as a control (iii). Western blot analysis of protein lysates from 3DO or GIRL19 treated with anti-CD3 mAb for the times indicated (C: untreated cells). The immunoblotting was performed using an mAb that recognizes the phosphorylated form of I-κBα (iv), or with a control antibody anti–I-κBα, phosphorylation-state independent (v). (B) Coimmunoprecipitation of p65/GILZ and p65/GST–I-κBα. In vitro–translated p65 (5 μL) was incubated overnight with GILZ (1 μg) or GILZ and GST–I-κBα (100 μg) and immunoprecipitated with anti-p65 antibody (3 μL). Immunoblotting was performed with antibody anti-GILZ (i, lane 1: nonimmunoprecipitated GILZ control; lane 2: p65 plus GILZ; lane 3: p65 plus GILZ plus GST–I-κBα; lane 4: p65 alone; lane 5: GILZ alone) or antibody anti-p65 (ii, lane 1: nonimmunoprecipitated p65 control; lane 2: p65 plus GILZ; lane 3: p65 plus GILZ plus GST–I-κBα; lane 4: p65 alone; lane 5: GILZ alone) or antibody anti–I-κBα (iii, lane 1: nonimmunoprecipitated GST–I-κBα control; lane 2: p65 plus GILZ; lane 3: p65 plus GILZ plus GST–I-κBα; lane 4: p65 plus GST–I-κBα; lane 5: GILZ plus GST-I-κBα).