title: Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.
abstract: BACKGROUND
  The 'phosphate-binding tag' (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.
  
  
  RESULTS
  We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.
  
  
  CONCLUSIONS
  We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.
authors.affiliation: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Hector N
authors.initials: HN
authors.lastname: Aguilar
authors.forename: Curtis N
authors.initials: CN
authors.lastname: Tracey
authors.forename: Siu Cheung F
authors.initials: SC
authors.lastname: Tsang
authors.forename: Justin M
authors.initials: JM
authors.lastname: McGinnis
authors.forename: Bryan F
authors.initials: BF
authors.lastname: Mitchell
mesh_terms: D001665:Binding Sites; D002457:Cell Extracts; D004151:Dipeptides / Q000494:pharmacology; D005260:Female; D006801:Humans; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010121:Oxytocin / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D015203:Reproducibility of Results; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014599:Uterus / Q000166:cytology*; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D004151:Dipeptides; D018994:Myosin Light Chains; D010703:Phorbol Esters; D010750:Phosphoproteins; C071482:calpeptin; D012694:Serine; D010121:Oxytocin; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase