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Total ROCK, RhoA GTPase and nNOS activity assays

JH Johanna L. Hannan
HM Hotaka Matsui
NS Nikolai A. Sopko
XL Xiaopu Liu
EW Emmanuel Weyne
MA Maarten Albersen
JW Joseph W. Watson
AH Ahmet Hoke
AB Arthur L. Burnett
TB Trinity J. Bivalacqua
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A separate group of MPGs were homogenized and total ROCK activity was analyzed in MPG lysate with of 0.1 mM adenosine triphosphate by a ROCK activity assay kit using peroxidase coupled anti-phospho-MYPT1 Thr696 monoclonal antibody (CyClex, Nagano, Japan). RhoA GTPase activity was assayed in MPG lysates by 96-well activity assays (Cytoskeleton, Denver, Colorado) according to manufacturer specifications. Total ROCK and RhoA GTPase activity are displayed as a percent of sham activity, as previously described8,12.

NOS activity in MPG tissues was assessed by radiolabeled L-arginine to L-citrulline conversion as previously described (Cayman, Chemical, Ann Arbor, MI, USA)8,39. Constitutive NOS activity measurements were performed in the presence of calcium and shown as a fold change from sham MPG values.

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