Fig. 5. DN Lck blocks SCF-mediated MAPK activation. H526 cells were stably transfected with the pTet-On plasmid followed by the pTRE plasmid containing either no insert (empty vector), the WT Lck cDNA, the Y505F Lck cDNA containing the constitutively activating mutation, or the DN Lck cDNA containing both the Y505F and K273R mutations. SCF-stimulated MAPK activation was assessed by immunoblotting with the activation-specific antibody in the presence or absence of doxycycline-induced expression of WT and mutant Lck. The extent of MAPK activation can also be estimated by assessing the mobility shift seen after staining with the pan-ERK antibody. A and B illustrate two representative experiments using two independently derived DN clones. In all, three independently derived clones expressing DN Lck were assayed in duplicate with consistent results. A, arrow, a background band intermittently detected between ERK 1 and ERK 2; the intensity of this band is independent of doxycycline or SCF treatment.