Tax induces JNK activity and subsequent phosphorylation of c-Jun.

(A) Total lysate of HepG2 cells transfected with a Tax expression vector (Tax) or an empty expression vector (control) was subjected to JNK kinase assay by using the GST-c-Jun_1-79 and to an immunoblot analysis probed with an anti–p-c-Jun, an anti-Tax, or an anti–α-actin antibody. (B) Nuclear extract from Tax expression vector (Tax) or an empty expression vector (control) transfected HepG2 cells were used for EMSA with an AP-1–specific probe, 50 M excess of non–radio-labeled AP-1 probe was added as competitor in 50 × molar excess (comp). (C,D) HepG2 cells were cotransfected with the (CAGA)₁₂-Luc reporter construct (2 μg) and the indicated combinations of Tax (5 μg) and/or c-Jun (5 μg), JNK (5 μg), dominant-negative JNK (JNK-(K-R); 5 μg), antisense c-Jun (c-Jun AS; 5 μg) expression vectors and were treated with or without TGF-β1. In the indicated condition, anisomycin (Aniso) was added 24 hours after transfection for 30 minutes before lysis. Error bars represent the variability of one of the experiments performed 3 times in duplicates. For each condition, a part of the lysate was subjected to immunoblot with a p-c-Jun antibody to assess the level of p-c-Jun.