ERK1/2 activity using AQT1076 selective sensor peptide (15 µM) with crude lysates (1-5 µg/well) from 7 cell models with or without cell stimulation and the inclusion of the selective ERK1/2 inhibitor SCH772984.

Reaction rates (RFU Corrected/pg of total lysate protein/min) were determined from the slopes using the linear portion of each progress curve. Values are the average of duplicate reactions +/- standard deviation. Green-shaded bars indicate stimulation of the cells before making lysates. Inhibitors confirming the selectivity of the assay were added to the lysate reaction.

AKT1/2/3 activity using AQT0982 selective sensor peptide (15 µM) with crude lysates (2 µg/well) from 13 cell lines with or without cell stimulation. Note that the signal with crude lysate from PDGF-treated NIH3T3 cells was completely inhibited with the inclusion of the selective AKT inhibitors Rizavasertib (A-443654) and Vevorisertib (ARQ 751) (1 µM of each; data not shown). 

Reaction rates (RFU Corrected/pg of total lysate protein/min) were determined from the slopes using the linear portion of each progress curve. Values are the average of duplicate reactions +/- standard deviation. Green-shaded bars indicate stimulation of the cells before making lysates. Inhibitors confirming the selectivity of the assay were added to the lysate reaction.

GSK3α/β Activity using AQT1211 selective sensor peptide (15 µM) with crude lysates (1.8 µg/well) from 9 cell models with or without stimulation and the inclusion of the selective GSK3α/β inhibitor LY2090314 (1 µM). Growth factor stimulation activates both ERK1/2 (via the RAF/MEK1/2/ERK1/2 pathway) and AKT (via the PI3K pathway), with varying levels of activation in different cell lines and response to various stimuli. With GSK3, its activation is the opposite of AKT, since AKT phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 inhibits GSK3 activity. 

Reaction rates (RFU Corrected/pg of total lysate protein/min) were determined from the slopes using the linear portion of each progress curve. Values are the average of duplicate reactions +/- standard deviation. Green-shaded bars indicate stimulation of the cells before making lysates. Inhibitors confirming the selectivity of the assay were added to the lysate reaction.