Expression of mitogen-activated protein kinase constitutive activators from lentivectors activates MAPKs in cell lines and dendritic cells. (A) Constitutive mitogen-activated protein kinase (MAPK) and IRF3 activators are shown as bars. Activating mutations are indicated within the bars. FLAG represents the FLAG epitope tag sequence at the amino-terminus of each activator. ΔNES indicates deletion of MEK1 nuclear export sequence. The structure of the dual lentivector is also shown: LTR, HIV-1 long terminal repeat; ψ, HIV packaging signal; RRE, HIV Rev response element; cPPT, HIV central polypurine tract; SFFV, spleen focus-forming promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; UBIQ, human ubiquitin promoter. ΔU3 represents the HIV-1 LTR with a deletion covering the U3 region, resulting in a SIN lentivector. (B) FLAG-specific immunoblots are shown of 293T cells transduced with the indicated lentivectors encoding activators. UT, extracts from untransduced cells. GFP, cells transduced with the lentivector encoding only GFP. MKK6, MEK1, represent mutants MKK6EE and MEK1 ΔNES ED, respectively. (C) Human 293T cells were transduced with lentivectors and immunoblots were performed from cellular extracts probing with the indicated antibodies on the left of each blot. Constitutive activators expressed from dual lentivectors are indicated on top. (D) Bone marrow (BM)–derived mouse dendritic cells (DCs) were transduced with the indicated dual lentivectors and incubated for 5 days at 37°C. FLAG-specific immunoblots were performed. (E) Protein extracts from transduced BM-derived mouse DCs were analyzed by immunoblot using the indicated antibodies on the left of each blot. Constitutive activators expressed from dual lentivectors are indicated on top of the blots. Positions of indicated MAPKs are shown by arrows on the left. Positions of specific MAPKs are indicated by arrows. Ab, antibodies; kDa, kilodaltons.