IE1-mediated activation of the A20 promoter in reporter assays. (A) Schematic representation of the NF-κB reporter genes used in reporter assays (see Materials and Methods). The binding sites for Sp1 and NF-κB are indicated. TK indicates the minimal core region of the HSV-1 TK promoter. (B) Via electroporation, HF cells were cotransfected with 0.5 μg of reporter plasmids containing the A20(NF-κB)-luciferase or the synthetic NF-κB-luciferase reporter gene (pNFκB-Luc) and 0.5 μg of plasmids expressing HA-IE1 or HA-IE2, as indicated. At 48 h, total cell lysates were prepared, and luciferase assays were performed. The levels of IE1, IE2, and β-actin are shown by immunoblotting with anti-HA and anti-β-actin Abs. (C) HF cells were cotransfected with an A20-luciferase reporter plasmid containing wild-type or mutant NF-κB sites [pA20(Wt)-Luc or pA20(mNF-κBs)-Luc] and plasmids expressing HA-IE1 or HA-IE2, and the luciferase assays and immunoblot analysis were performed. (D and E) Control HF cells (MIN) or cells expressing IE1 (MIN-IE1) were produced by retroviral transduction. Total cell lysates were prepared and immunoblot analysis were performed with mouse MAbs for A20, IE1 (810R) or β-actin (D). Cells were fixed in methanol and double-alabel IFA was performed with anti-A20 and anti-IE1 Abs. Rhodamine/Red X-coupled anti-mouse IgG and FITC-conjugated anti-rabbit IgG were used for visualization (E).