GILZ inhibits NF-κB binding to its DNA motif in an EMSA assay.

(A) EMSA assay performed using nuclear extract from untreated (lanes 2-5) and anti-CD3–treated (2 hours, lanes 6-9) 3DO cells (lanes 3, 7) and empty vector– (PV6, lanes 2, 6) or GILZ-transfected (ST7, lanes 4, 8; GIRL19, lanes 5, 9) clones. Lane 1: probe alone. (B) Nuclear extract from anti-CD3–treated 3DO cells, alone (lane 3) or added with GST-GILZ fusion protein (lane 4), or buffer used for GST-GILZ preparation alone (lane 5), or GST-P56^lck fusion protein (lane 6), or GST alone (lane 7) as controls. Lane 1: probe alone. Lane 2: GILZ-GST alone. (C) Nuclear extract from untreated (lanes 1, 2) or anti-CD3–stimulated (lanes 3-5), empty-vector– (PV6, lanes 1, 3, 4), or GILZ-transfected (ST7, lanes 2, 5) cells. Lane 3: control with nuclear extract from anti-CD3–treated empty vector–transfected cells plus competitor cold probe. Lane 6: labeled probe alone. (D) Nuclear extract of untreated (lanes 1, 5) or anti-CD3–treated (lanes 2-4, 6, 7) PV6 cells. Nuclear extract plus anti-p65 antibody (lane 3) or control antibody (lane 4). Nuclear extract plus GILZ-GST fusion protein (lanes 5, 6). Nuclear extract plus anti-p65 and GILZ-GST fusion protein (lane 7). Lane 8: GILZ-GST fusion protein alone. Lane 9: probe alone. (E) Nuclear extract of untreated (lane 2) or anti-CD3–treated PV6 (lanes 3-6) cells. Nuclear extract plus anti-p52 antibody (lanes 5, 6) or control antibody (lane 4). Nuclear extract plus anti-p52 and GILZ-GST fusion protein (lane 6). Lane 1: probe alone. (F,G) EMSA assay performed using as probe NF-AT and OCT-1, respectively. Nuclear extracts from untreated (lanes 2-5) or anti-CD3–treated (lanes 6-9) 3DO,3 7 PV6 (lanes 2, 6), ST7 (lanes 4, 8), and GIRL19 (lanes 5, 9). Lane 1: probe alone.