Validation of non-cleavable MKKs. (A) Non-cleavable MKK mutants or empty vector plasmid were transiently transfected into HeLa cells and expressed for 24 h followed by 24 h of LT treatment. Cells were then stimulated for 15 min with PMA (100 ng/ml) or TNFα (25 µg/ml) to activate MKK/MAPK pathways. Endogenous MKK expression; exogenous, non-cleavable MKK expression; LT-mediated MKK cleavage; phosphorylation of MAP kinases Erk, p38 or JNK; and total Erk, p38 and JNK expression are visualized by immunoblot as indicated. (B) Lentiviral expression of non-cleavable MKK pairs (MKK1/2, MKK3/6, MKK4/7) together with EGFP and of EGFP alone in SALE cells (see Methods). Lysates of the four SALE cell lines were probed by immunoblot with MKK specific and GFP antibodies. (1.45 MB TIF)