Fig. 6. Activation of PKB, S6K and PFK2 in heart by insulin. Mice were fasted overnight and injected with either saline for 10–20 min (for the 0 time point control) or 1 mU/g of insulin for the indicated times. The heart was then rapidly extracted and frozen in liquid nitrogen. PKB (A) or S6K (B) were immunoprecipitated from cardiac extract and the activity determined using a quantitative peptide phosphorylation assay. Each point represents the mean activity ± SD of three different hearts with each assayed in triplicate. (C and D) As above except that cell lysates form the indicated hearts were immunoblotted with the indicated antibodies. Each lane represents a different mouse PFK-2 activity (E) and fructose-2,6-bisphosphate (Fru-2,6-P2) levels (F) were measured as described in the Materials and methods. Each point represents the mean activity ± SD of three to five different hearts with each assayed in duplicate.