Figure 6.
The fiveplex kinase assay measures TNF- and insulin-induced activation patterns that correlate with conventional kinase data and show better dynamic range than chelation-enhanced fluorescence (ChEF)-based peptide-phosphorylation reporters40–42 applied to cell extracts. Data by phosphokinase immunoblot (p-IB) and radioactive kinase assay (KA) from Gaudet et al.39 are shown for (a) Akt, (b) IKK, (c) JNK, (d) MEK, and (e) MK2 and compared with (f–j) fiveplex KA measurements and (k–o) ChEF reporter activity measurements from HT29 cells stimulated with 100 ng/ml TNF or 500 ng/ml insulin for the indicated time points. Data are shown as the mean ± s.d. of n = 3–6 biological replicates normalized as described39 or to the mean HT29 0-hr time point.
