| Assay Method Information | |
| | Inhibition of KRas G12D-mediated Phosphorylation of ERK |
| Description: | This Example illustrates that exemplary compounds of the present invention inhibit the phosphorylation of ERK downstream of KRAS G12D. AGS cells (ATCC CRL-1739) expressing G12D were grown in DMEM medium supplemented with 10% fetal bovine serum, 10 mM HEPES, and Penicillin/Streptomycin. Cells were plated in tissue culture treated 96 well plates at a density of 40,000 cells/well and allowed to attach for 12-14 hours. Diluted compounds were then added in a final concentration of 0.5% DMSO. After 3 hours, the medium was removed, 150 μL of 4.0% formaldehyde was added and the plates incubated at room temperature for 20 minutes. The plates were washed with PBS, and permeabilized with 150 μL of ice cold 100% methanol for 10 minutes. Non-specific antibody binding to the plates was blocked using 100 L Licor blocking buffer (Li-Cor Biotechnology, Lincoln Nebr.) for 1 hour at room temperature. The amount of phospho-ERK was determined using an antibody specific for the phosphorylated form of ERK and compared to the amount of GAPDH. Primary antibodies used for the detection were added as follos: Phospho-ERK(Cell Signaling cs-9101) diluted 1:500 and GAPDH(Millipore MAB374) diluted 1:5000 in Licor block+0.05% Tween 20. The plates were incubated for 2 hours at room temperature. The plates were washed with PBS+0.05% Tween 20. Secondary antibodies used to visualize primary antibodies were added as follows: Anti-rabbit-680 diluted 1:1000 and Anti-mouse-800 diluted 1:1000 both in Licor block+0.05% TweeN20, and were incubated for 1 hour at room temperature. The plates were washed with PBS +0.05% Tween 20. A 100 μL aliquot of PBS was added to each well and the plates were read on a Li-Cor Odyssey CLX plate reader. |
| Affinity data for this assay | |
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