Journal: The Journal of Biological Chemistry
Article Title: Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I
doi: 10.1074/jbc.RA119.011945
Figure Lengend Snippet: In situ phosphorylation of cTnI and cRLC by cMLCK in troponin-exchanged rat ventricular trabeculae. A , recombinant cardiac troponin exchange efficiency in ventricular trabeculae, determined by Western blotting against cTnT. Endogenous native rat cTnT ( left ) migrates faster in SDS-PAGE than recombinant human cTnT ( right ), allowing estimation of the cTn exchange efficiency into rat ventricular trabeculae ( center ). Mean ± S.E., n = 3. B , low-molecular-weight portion of SDS-PAGE of ventricular trabeculae treated without (−) and with (+) protein kinase inhibitors (H-89 and staurosporine). Gels were stained with phospho-specific Pro-Q Diamond or SYPRO total protein stain. Densitometric analysis for cTnI is shown on the right . Means ± S.E., n = 6. C and D , rat cTn-exchanged ( C ) or human cTn-exchanged ( D ) demembranated ventricular trabeculae were incubated without (− cMLCK , time-matched control) or with cMLCK (+ cMLCK ), and phosphorylation levels were determined by Phos-tag TM SDS-PAGE, followed by Western blotting against cTnI ( top ) and cRLC ( bottom ). Unphosphorylated human cTnI (indicated by 0P' ) migrates faster than unphosphorylated rat cTnI ( 0P ). TnI degradation products are labeled accordingly ( Deg ). Ponceau stains for endogenous rat ( rcTnT ) and exogenous human cardiac troponin T ( hcTnT ) are shown in the center panels. E , densitometric analysis of cTnI ( top ) and cRLC phosphorylation ( bottom ) levels before (−) and after cMLCK (+) incubation in C and D . Means ± S.E., n = 3–6. Statistical significance of differences was assessed with an unpaired, two-tailed Student's t test. ns , not significant; *, p < 0.05; ***, p < 0.001.
Article Snippet: Cardiac troponins were gel-filtered into cMLCK assay buffer (50 mmol/liter HEPES, 50 mmol/liter NaCl, 2 mmol/liter MgCl 2 , 1 mmol/liter CaCl 2 , and 1 mmol/liter DTT) via NAP5 columns (GE Healthcare).
Techniques: In Situ, Recombinant, Western Blot, SDS Page, Molecular Weight, Staining, Incubation, Labeling, Two Tailed Test
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