Quantification of rapid myosin regulatory light chain phosphorylation using high-throughput in-cell western assays: Comparison to western immunoblots

PLoS ONE

Volumn 5, Issue 4, 2010, Pages

  Aguilar, Hector N ^a   Zielnik, Barbara ^a   Tracey, Curtis N ^a   Mitchell, Bryan F ^a  

^a UNIVERSITY OF ALBERTA   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

MYOSIN LIGHT CHAIN; PHOSPHOPROTEIN;

ACCURACY; ANALYTIC METHOD; ARTICLE; CONTROLLED STUDY; HIGH THROUGHPUT SCREENING; HUMAN; HUMAN CELL; IN CELL WESTERN TECHNIQUE; INTERMETHOD COMPARISON; MUSCLE CELL; MYOMETRIUM; PROTEIN ANALYSIS; PROTEIN PHOSPHORYLATION; PROTEIN PURIFICATION; PROTEIN TARGETING; SENSITIVITY AND SPECIFICITY; VALIDITY; WESTERN BLOTTING; CELL CULTURE; CHEMISTRY; COMPARATIVE STUDY; CYTOLOGY; EVALUATION; FEMALE; IMMUNOASSAY; INFRARED SPECTROPHOTOMETRY; METHODOLOGY; PHOSPHORYLATION; UTERUS;

BLOTTING, WESTERN; CELLS, CULTURED; CYTOLOGICAL TECHNIQUES; FEMALE; HUMANS; IMMUNOASSAY; MUSCLE CELLS; MYOSIN LIGHT CHAINS; PHOSPHOPROTEINS; PHOSPHORYLATION; SENSITIVITY AND SPECIFICITY; SPECTROPHOTOMETRY, INFRARED; UTERUS;

EID: 77956322925     PISSN: None     EISSN: 19326203     Source Type: Journal    
DOI: 10.1371/journal.pone.0009965     Document Type: Article

References (31)

1. Weldon S, Ambroz K, Schutz-Geschwender A, Olive DM (2008) Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis. Anal Biochem 375: 156-158.
2. Wang YV, Wade M, Wong E, Li YC, Rodewald LW, et al. (2007) Quantitative analyses reveal the importance of regulated Hdmx degradation for p53 activation. Proc Natl Acad Sci U S A 104: 12365-12370.
3. Jungblut P, Eckerskorn C, Lottspeich F, Klose J (1990) Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources. Electrophoresis 11: 581-588.
4. Tovey ER, Baldo BA (1987) Comparison of semi-dry and conventional tankbuffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis 8: 384-387.
5. Selkirk JV, Nottebaum LM, Ford IC, Santos M, Malany S, et al. (2006) A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation. J Biomol Screen 11: 351-358.
6. Kumar N, Afeyan R, Kim HD, Lauffenburger DA (2008) Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2- overexpressing mammary epithelial cells. Mol Pharmacol 73: 1668-1678.
7. Coevoets R, Arican S, Hoogeveen-Westerveld M, Simons E, van den Ouweland A, et al. (2009) A reliable cell-based assay for testing unclassified TSC2 gene variants. Eur J Hum Genet 17: 301-310.
8. Bond D, Primrose DA, Foley E (2008) Quantitative evaluation of signaling events in Drosophila s2 cells. Biol Proced Online 10: 20-28.
9. Schroter T, Griffin E, Weiser A, Feng Y, LoGrasso P (2008) Detection of myosin light chain phosphorylation-a cell-based assay for screening Rho-kinase inhibitors. Biochem Biophys Res Commun 374: 356-360.
10. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T (2006) Phosphatebinding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5: 749-757.
11. Takeya K, Loutzenhiser K, Shiraishi M, Loutzenhiser R, Walsh MP (2008) A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles. Am J Physiol Renal Physiol 294: F1487-1492.
12. Smith PJ, Wiltshire M, Davies S, Patterson LH, Hoy T (1999) A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry. J Immunol Methods 229: 131-139.
13. Kim HR, Appel S, Vetterkind S, Gangopadhyay SS, Morgan KG (2008) Smooth muscle signalling pathways in health and disease. J Cell Mol Med 12: 2165-2180.
14. Takashima S (2009) Phosphorylation of myosin regulatory light chain by myosin light chain kinase, and muscle contraction. Circ J 73: 208-213.
15. Hunter T (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80: 225-236.
16. Bhadriraju K, Elliott JT, Nguyen M, Plant AL (2007) Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy. BMC Cell Biol 8: 43.
17. Word RA, Stull JT, Casey ML, Kamm KE (1993) Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women. J Clin Invest 92: 29-37.
18. Zhou XB, Lutz S, Steffens F, Korth M, Wieland T (2007) Oxytocin receptors differentially signal via Gq and Gi proteins in pregnant and nonpregnant rat uterine myocytes: implications for myometrial contractility. Mol Endocrinol 21: 740-752.
19. Sharma SK, Carew TJ (2002) Inclusion of phosphatase inhibitors during Western blotting enhances signal detection with phospho-specific antibodies. Anal Biochem 307: 187-189.
20. Abeyrathne PD, Lam JS (2007) Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots. Can J Microbiol 53: 526-532.
21. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76: 4350-4354.
22. Bolt MW, Mahoney PA (1997) High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 247: 185-192.
23. Egger D, Bienz K (1994) Protein (western) blotting. Mol Biotechnol 1: 289-305.
24. DenHollander N, Befus D (1989) Loss of antigens from immunoblotting membranes. J Immunol Methods 122: 129-135.
25. Tovey ER, Baldo BA (1989) Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting. J Biochem Biophys Methods 19: 169-183.
26. Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ (2008) The use of total protein stains as loading controls: an alternative to high-abundance singleprotein controls in semi-quantitative immunoblotting. J Neurosci Methods 172: 250-254.
27. Mandell JW (2003) Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol 163: 1687-1698.
28. Arnon R, Van Regenmortel MH (1992) Structural basis of antigenic specificity and design of new vaccines. FASEB J 6: 3265-3274.
29. Weber LP, Van Lierop JE, Walsh MP (1999) Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J Physiol 516 (Pt 3): 805-824.
30. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
31. Lundholt BK, Scudder KM, Pagliaro L (2003) A simple technique for reducing edge effect in cell-based assays. J Biomol Screen 8: 566-570.