Western blot analysis of extracts from 293 cells, untreated or treated with the ROCK inhibitor Y27632 (2 minutes at 10 μM) using Phospho-MYPT1 (Thr696) Antibody or total MYPT1 Antibody #2634. Phospho-MYPT1 (Thr696) Antibody was pre-incubated as indicated with phospho-MYPT1 (Thr696) peptide or nonphospho-MYPT1 (Thr696) peptide.
|REACTIVITY||H M R Mk|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
Phospho-MYPT1 (Thr696) Antibody detects endogenous levels of MYPT1 only when phosphorylated at Thr696. The antibody may cross-react with the phospho-MYPT2 (Thr 646) due to high sequence homology.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surroundiing Thr696 of human MYPT1. Antibodies are purified using protein A and peptide affinity chromatography.
Protein phosphatase 1 (PP1) is a ubiquitous eukaryotic protein serine/threonine phosphatase involved in the regulation of various cell functions. Substrate specificity is determined by the binding of a regulatory subunit to the PP1 catalytic subunit (PP1c). It is estimated that over fifty different regulatory subunits exist (1).
The myosin phosphatase holoenzyme is composed of three subunits: PP1c, a targeting/regulatory subunit (MYPT/myosin-binding subunit of myosin phosphatase), and a 20 kDa subunit of unknown function (M20). MYPT binding to PP1cδ alters the conformation of the catalytic cleft and increases enzyme activity and specificity (2). Two MYPT isoforms that are 61% identical have been described. MYPT1 is widely expressed, while MYPT2 expression appears to be exclusive to heart and brain (3). Related family members include MBS85, MYPT3, and TIMAP (4).
Myosin phosphatase regulates the interaction of actin and myosin in response to signaling through the small GTPase Rho. Rho activity inhibits myosin phosphatase via Rho-associated kinase (ROCK). Phosphorylation of MYPT1 at Thr696 and Thr853 results in phosphatase inhibition and cytoskeletal reorganization (5,6).
Explore pathways + proteins related to this product.
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