The Right Tools for Many Applications – Keep Your Research on the Fast Track
Effective Screening of Inhibitors for Influence on Proliferation and Survival of Mouse Neural Stem Cells.
Rapid Screening of Receptor Tyrosine Kinase (RTK) Inhibition Using InhibitorSelect™ Inhibitor Library I and WideScreen™ RTK pTyr and Total 7-plex Assay Kits on a Luminex® xMAP® Platform.
InhibitorSelect 96-Well Protein Kinase Inhibitor Libiraries I & II (160 inhibitors; Cat. No. 539744 and 539745) were screened for influence on the proliferation and survival of the mouse neural stem cells (mNS) in an ATP bioluminescence cell viability assay. mNS cells were plated at 3,000 cells/well and viability assay performed after 2 days of incubation. Each compound was tested in each of four growth factor backgrounds:
Click image to enlarge.
Results for 20 inhibitors are presented as mean data (of n-4 wells per condition) with error bars indicating standard error of the mean (SEM). The presence of inhibitor K-252a, Nocardiospsis sp. (Cat. No. 420297) alone in the culture media resulted in a 10-fold mNS cell viability.
- (A) No GFs – No Growth Factors (to identify survival/ proliferation factors)
- (B) Sub EGF – Sub-optimal EGF (to identify inhibitors/ potentiators) 20 pg/mL EGF
- (C) Sub FGF2 – Suboptimal FGF2 (to identify inhibitors/ potentiators) 500 pg/mL FGF2
- (D) Max GFs – Maximal EGF + FGF2 (to identify inhibitors/ potentiators) 20 ng/mL EGF + 20 ng/mL FGF2
Data courtesy of Donna McLaren, Stem Cell Sciences, Cambridge, UK
Over twenty kinase inhibitors were rapidly screened for their ability to inhibit RTK activation using the InhibitorSelect 96-well Inhibitor Library I (Cat. No. 539744). Cell lines were stimulated with the select growth factors or mitogens. Following stimulation, the activation and inhibition of several TRKs (EGFR, HER2, HGFR, IGF1R, PDGFR, TIE2, and VEGFR2), were measured using the WideScreen RTK pTyr 7-plex Assay (Cat. No. 71943). RTK phosphorylation levels were normalized against total RTK concentrations using WideScreen RTK Total 7-plex Assay (Cat. No. 71942). Representative data for VEGFR2 are shown.
Click image to enlarge.
Maximize Your Results and Minimize Your Screening Cost
Characteristics of three Akt inhibitors included in InhibitorSelect Libraries are shown to demonstrate the diversity and quality of inhibitors provided.
Akt Inhibitor V, Triciribine (Cat. No. 124012
Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2 (Cat. No. 124018
Akt Inhibitor X (Cat. No. 124020
- Cameron AJ, Escribano C, Saurin AT, Kostelecky B, Parker PJ. PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity. Nature Structural & Molecular Biology 16, 624 - 630 (2009).
- Chou TF, Deshaies RJ. Quantitative Cell-based Protein Degradation Assays to Identify and Classify Drugs That Target the Ubiquitin-Proteasome System JBC 286, 16546-16554 (2011)
- Farkas T, Høyer-Hansen M, Jäättelä, M. Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux. Autophagy 5 (7), 1018-1025 (2009)
- Gronenberg LS, Kahne D. Development of an Activity Assay for Discovery of Inhibitors of Lipopolysaccharide Transport. J. Am. Chem. Soc. 132 (8), pp 2518–2519 (2010).
- Hooper S, Gaggioli C, Sahai E. A chemical biology screen reveals a role for Rab21-mediated control of actomyosin contractility in fibroblast-driven cancer invasion. British J of Cancer 102, 392-402 (2010)
- Johnson DL, Stone CB, Bulir DC, Coombes BK, Mahony JB. A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication. BMC Microbiology 9:218 (2009).
- Lee AJ, Endesfelder D, Rowan AJ, Walther A, Birkbak NJ, Futreal PA, Downward J, Szallasi Z, Tomlinson IP, Howell M. Chromosomal instability confers intrinsic multidrug resistance. Cancer Res. 71 (5), 1858-1870 (2011)
- Li T, Liu D, Wang Z. Screening Kinase Inhibitors with a Microarray-Based Fluorescent and Resonance Light Scattering Assay. Anal. Chem. 82, 3067–3072 (2010)