HTRF® phospho-assays reveal subtle drug-induced effects in tumor xenografts. A method of choice beyond Western blot

J. Vallaghé1, D. Monchiet1, N. Costy1, T. Chardès2, C. Larbouret2, E. Trinquet1, F. Charrier-Savournin1

2015

1 Cisbio Bioassays, Codolet, France | 2 Institut de Recherche en Cancérologie de Montpellier, INSERM U896, Université Montpellier 1, Institut régional du Cancer Montpellier / Val d’Aurelle, France

SLAS 2015

Cell signaling pathways involving phospho-proteins are often over-activated in cancers, leading to aberrant cell proliferation and survival. Therefore, protein phosphorylation is frequently assessed while developing novel anti-tumor therapeutics, such as tyrosine kinase inhibitors (TKI) or monoclonal antibodies. In preclinical cancer studies, human tumor xenograft-bearing mice are routinely used as predictive models to evaluate the in vivo efficacy of such anticancer drugs. Western blot is the standard technique for analyzing proteins in tumor lysates, but it is time- and labor-intensive.
This poster compares the use of HTRF phospho-assays with traditional Western blot for the analysis of AKT and ERK1/2 in human pancreatic tumor xenografts, after mouse treatment with the anti-EGFR TKI erlotinib. The data demonstrates that HTRF is a more convenient, accurate and sample-saving method than Western blot for assessing protein phosphorylation in tumor xenografts.

Phosphorylated proteins

Phospho-4EBP1 (Thr37/46) Cellular Assay KitPhospho-ACC (Ser79) Cellular Assay KitPhospho-AMPK (Thr172) & Total AMPK Cellular Assay Kits Phospho-ATF2 (Thr71) Cellular Assay KitPhospho-β-Catenin (Thr41/Ser37/Ser33) & Total β-Catenin Cellular Assay Kits Phospho-Bad (Ser112) Cellular Assay Kit Phospho-c-Jun (Ser63) Cellular Assay KitPhospho-c-RAF (Ser338) Cellular Assay KitPhospho-EGFR (pan), Phospho-EGFR (Tyr1068) & Total EGFR Cellular Assay KitsPhospho-EIF4E (Ser209) Cellular Assay Kit Phospho-ERK (Thr202/Tyr204), Advanced phospho-ERK (Thr202/Tyr204), and Total ERK1/2 Cellular Assay KitsPhospho-GSK3β (Ser9), Phospho-GSK3α (Ser21), Total GSK3β & Total GSK3α Cellular Assay KitsPhospho-H2AX (Ser139) Cellular Assay KitPhospho-HER2 (Tyr1221/1222) & Total HER2 Cellular Assay KitsPhospho-HER3 (Tyr1289) & Total HER3 Cellular Assay KitsPhospho-Histone H3 (Ser10), Phospho-Histone H3 (Thr3) & Total Histone H3 Cellular Assay KitsPhospho-IKKβ (Ser177/181) & Total IKKβ Cellular Assay Kit Phospho-JNK (Thr183 / Tyr185) Cellular Assay Kit Phospho-MEK1 (Ser218/222), Phospho-MEK1/2 (Ser218/222), Phospho-MEK1 (Ser298) & Total MEK1 Cellular Assay KitsPhospho-mTOR (Ser2448) Cellular Assay KitPhospho-NFkB (Ser536) & Total NFkB Cellular Assay KitsPhospho-p38 MAPK (Thr180/Tyr182) Cellular Assay KitPhospho-P70S6K (Thr389) Cellular Assay Kit Phospho-S6RP (Ser235/236) Cellular Assay Kit Phospho-STAT3 (Tyr705) & Total STAT3 Cellular Assay KitsPhospho-STAT5 (Tyr694) Cellular Assay KitPhospho-YAP (Ser127) & Total YAP Cellular Assay KitsPhospho-SMAD1 (Ser463/465) & Total SMAD1 Cellular Assay KitsPhospho-p53 (Ser15) & Total p53 Cellular Assay KitsPhospho- & total AKT 1/2/3, Phospho- & total pan AKT Cellular Assay KitsPhospho-FGFR2 (Tyr653/654) & Total FGFR2 Cellular Assay KitsPhospho-MNK (Thr197/202) Cellular Assay KitPhospho-ULK1 (Ser757 mouse / Ser758 human) Assay KitTotal IRS2 Cellular Assay KitPhospho-SLP-76 (Ser376) & total SLP-76 Cellular Assay KitsPhospho-FoxO1 (Ser256) and Total FoxO1 Assay KitsPhospho-c-Src (Tyr419) and Total c-Src Assay KitsPhospho-IRF3 (Ser386) Cellular Assay KitPhospho-SMAD3 (Ser423/425) & Total SMAD3 Cellular Assay KitsPhospho-EIF2 alpha (Ser52) & Total EIF2 alpha Cellular Assay KitsAlpha-Tubulin Housekeeping Cellular KitSTAT1 phospho (Tyr701) & total assaysPhospho-TBK1 (Ser172) & Total TBK1 Assay KitsPhospho-PDGFR beta (Tyr751) Cellular Assay Kit