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Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

Aguilar HN, Tracey CN, Tsang SC, McGinnis JM, Mitchell BF - PLoS ONE (2011)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

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.

Methodology/principal findings: 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.

Conclusion/significance: 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.

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Quantification of RLC phospho-state distribution.Panels A-D correspond to WB data derived using an Ab directed toward the C-terminus of RLC. A. Representative WBs demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. B. Quantification of bands identified in panel A (untreated; n = 12, g-H; n = 5, Calp; n = 8, OT; n = 7). The signals derived from 0pRLCT, 1pRLCT, and 2pRLCT are expressed as the proportion of their sum total within each lane. C. Absolute magnitude of the difference in each phospho-state caused by the treatments in comparison to the corresponding vehicle (‘+’ indicates enhancement relative to vehicle, ‘−’ indicates diminution relative to vehicle). D. Vehicle-corrected distribution data obtained by combining the data in panel C to the untreated group distribution. g-H (glycyl-H-1152); ROK inhibitor (1 µM). Calp (calpeptin); rhoA activator (0.5 mU/mL). OT; oxytocin (100 nM). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 1.
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pone-0020903-g003: Quantification of RLC phospho-state distribution.Panels A-D correspond to WB data derived using an Ab directed toward the C-terminus of RLC. A. Representative WBs demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. B. Quantification of bands identified in panel A (untreated; n = 12, g-H; n = 5, Calp; n = 8, OT; n = 7). The signals derived from 0pRLCT, 1pRLCT, and 2pRLCT are expressed as the proportion of their sum total within each lane. C. Absolute magnitude of the difference in each phospho-state caused by the treatments in comparison to the corresponding vehicle (‘+’ indicates enhancement relative to vehicle, ‘−’ indicates diminution relative to vehicle). D. Vehicle-corrected distribution data obtained by combining the data in panel C to the untreated group distribution. g-H (glycyl-H-1152); ROK inhibitor (1 µM). Calp (calpeptin); rhoA activator (0.5 mU/mL). OT; oxytocin (100 nM). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 1.

Mentions: This analysis has two primary goals: 1) isolating the effect of the treatment of interest from any vehicle-induced change in the phospho-state distribution, and 2) facilitating comparisons across treatments by expressing them relative to a common reference point (untreated distribution). Figure 3A shows representative WBs obtained for the treatment groups described. Distribution data were obtained by expressing the signal integrated intensity at each band position as a fraction of their sum total (e.g. [signal 0pRLCT]/([signal 0pRLCT]+[signal 1pRLCT]+[signal 2pRLCT]), etc.), and averaging these proportions for n = 5–8 samples. This ‘sum method’ amounts to performing an ‘in-lane’ normalization, in place of that commonly achieved by using a loading control. We propose that this ‘in-lane normalization’ method has several advantages that are discussed in a subsequent section. Note that the WBs have been intensified for all figures so that all three bands are visible, where possible. This is achieved by restricting the upper and lower boundaries of pixel intensity that are shown by the quantification software. The quantitative estimates of band intensity are completely independent of these manipulations. The blots presented here did not exhibit pixel saturation, and therefore have not exceeded the upper limit of the scanner in detecting higher protein loads.


Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

Aguilar HN, Tracey CN, Tsang SC, McGinnis JM, Mitchell BF - PLoS ONE (2011)

Quantification of RLC phospho-state distribution.Panels A-D correspond to WB data derived using an Ab directed toward the C-terminus of RLC. A. Representative WBs demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. B. Quantification of bands identified in panel A (untreated; n = 12, g-H; n = 5, Calp; n = 8, OT; n = 7). The signals derived from 0pRLCT, 1pRLCT, and 2pRLCT are expressed as the proportion of their sum total within each lane. C. Absolute magnitude of the difference in each phospho-state caused by the treatments in comparison to the corresponding vehicle (‘+’ indicates enhancement relative to vehicle, ‘−’ indicates diminution relative to vehicle). D. Vehicle-corrected distribution data obtained by combining the data in panel C to the untreated group distribution. g-H (glycyl-H-1152); ROK inhibitor (1 µM). Calp (calpeptin); rhoA activator (0.5 mU/mL). OT; oxytocin (100 nM). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111472&req=5

pone-0020903-g003: Quantification of RLC phospho-state distribution.Panels A-D correspond to WB data derived using an Ab directed toward the C-terminus of RLC. A. Representative WBs demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. B. Quantification of bands identified in panel A (untreated; n = 12, g-H; n = 5, Calp; n = 8, OT; n = 7). The signals derived from 0pRLCT, 1pRLCT, and 2pRLCT are expressed as the proportion of their sum total within each lane. C. Absolute magnitude of the difference in each phospho-state caused by the treatments in comparison to the corresponding vehicle (‘+’ indicates enhancement relative to vehicle, ‘−’ indicates diminution relative to vehicle). D. Vehicle-corrected distribution data obtained by combining the data in panel C to the untreated group distribution. g-H (glycyl-H-1152); ROK inhibitor (1 µM). Calp (calpeptin); rhoA activator (0.5 mU/mL). OT; oxytocin (100 nM). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 1.
Mentions: This analysis has two primary goals: 1) isolating the effect of the treatment of interest from any vehicle-induced change in the phospho-state distribution, and 2) facilitating comparisons across treatments by expressing them relative to a common reference point (untreated distribution). Figure 3A shows representative WBs obtained for the treatment groups described. Distribution data were obtained by expressing the signal integrated intensity at each band position as a fraction of their sum total (e.g. [signal 0pRLCT]/([signal 0pRLCT]+[signal 1pRLCT]+[signal 2pRLCT]), etc.), and averaging these proportions for n = 5–8 samples. This ‘sum method’ amounts to performing an ‘in-lane’ normalization, in place of that commonly achieved by using a loading control. We propose that this ‘in-lane normalization’ method has several advantages that are discussed in a subsequent section. Note that the WBs have been intensified for all figures so that all three bands are visible, where possible. This is achieved by restricting the upper and lower boundaries of pixel intensity that are shown by the quantification software. The quantitative estimates of band intensity are completely independent of these manipulations. The blots presented here did not exhibit pixel saturation, and therefore have not exceeded the upper limit of the scanner in detecting higher protein loads.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

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.

Methodology/principal findings: 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.

Conclusion/significance: 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.

Show MeSH
Related in: MedlinePlus