Fig. 4. Overexpression of Lck decreases the sensitivity of SCF-mediated MAPK activation to PP1. H526 cells were stably transfected with the pTet-On plasmid followed by the pTRE plasmid containing either no insert (empty vector) or the murine WT Lck cDNA. Cultures of cells containing pTRE and the WT Lck expression plasmid were treated with doxycycline for 24 h; a second culture of WT Lck-containing cells was not induced with doxycycline. All of the cultures were incubated in serum-free media overnight and were left unstimulated or were stimulated for 5 min with 100 ng/ml SCF after a 30-min preincubation with the indicated concentrations of PP1. Each culture was split for making whole-cell lysates (for analysis of ERK activation and Lck expression) and for Kit IP. Far-right column, the antibodies that were used to stain each immunoblot. Whole-cell lysates were initially stained for active ERK, were stripped, and were then stained with the pan-ERK antibody. Immunoprecipitates were first stained for phosphotyrosine followed by Kit. Only the Kit IP from the induced WT Lck culture is shown; others showed a pattern of Kit inhibition similar to that illustrated here and in Fig. 3 . Far-left column, the relative levels of Lck expression in each culture. Persistence of ERK activation above basal levels (levels in cells not treated with SCF) in the PP1-treated WT Lck + Dox culture indicates that Lck overexpression induces partial resistance to PP1. Densitometric analysis revealed the following normalized percentage of ERK activity in cells treated with SCF in the presence of 10 µM PP1 relative to basal ERK activity (last lane ÷ first lane x 100): pTRE + Dox, 61%; WT Lck - Dox, 70%; WT Lck + Dox, 257%. Dox, doxycycline.