Constitutive activation of ERK and Akt/PKB pathways in HT29 and LoVo cells. (a) Equal amounts of HT29 and LoVo cell extracts (50 μg) were examined for activation of ERKs and Akt/PKB through detection of phosphorylated ERK1/2 and Akt by immunoblot analysis. Blots were normalized to levels of total ERK1/2 and Akt and the data shown represent duplicate experiments. (b) ERK and Akt/PKB kinase assays. Equal amounts of HT29 and LoVo cell lysates (200 μg) were immunoprecipitated using monospecific phospho-ERK1/2 or phospho-Akt Ab’s. The immunoprecipitate was then incubated with ELK-1 or GSK-3 fusion proteins in the presence of ATP and kinase activity was detected by immunoblotting using Ab’s against phospho-ELK-1 or phospho-GSK-3. Blots were quantitated digitally and kinase activity is presented as density units. The results shown are the average of duplicate experiments.