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Lysates prepared from COS cells transfected with muscle MLCK left unstimulated, or phosphorylated in vitro by PKA, were resolved by SDS-PAGE on a 4-12% polyacrylamide gel and transferred to PVDF. Membranes were left untreated or treated with lambda phophatase, blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with MLCK [pS1760] antibody for two hours at room temperature in 3% BSA-TBST buffer, following prior incubation with: no peptide, the non-phosphopeptide corresponding to the immunogen, a generic phosphoserine-containing peptide, or the phosphopeptide immunogen. After washing, membranes were incubated with goat F (ab"e;) 2 anti-rabbit IgG HRP conjugate (Cat. no. ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that PKA induces the phosphorylation of MLCK at serine 1760. The data also show that only the phosphopeptide corresponding to MLCK [pS1760] blocks the signal and that phosphatase stripping also eliminates the signal, verifying that the antibody is indeed phosphorylation site-specific. (The transfected cell lysates were a generous gift from Dr. James Stull [UTSW]).
|Tested species reactivity||Bovine, Mouse, Rabbit|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human myosin light chain kinase (MLCK) that contains serine 1760. The sequence is conserved in human, mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
MLCK, a member of the Ser/Thr protein kinase family, is a calcium/calmodulin-dependent enzyme responsible for smooth muscle contraction via phosphorylation of a specific serine in the N-terminus of myosin light chains (MLC), an event that facilitates myosin interaction with actin filaments. It is a central determinant in the development of vascular permeability and tissue edema formation. In the nervous system it has been shown to control the growth initiation of astrocytic processes in culture and to participate in transmitter release at synapses formed between cultured sympathetic ganglion cells. MLCK acts as a critical participant in signaling sequences that result in fibroblast apoptosis. Smooth muscle and non-muscle isozymes are expressed in a wide variety of adult and fetal tissues and in cultured endothelium with qualitative expression appearing to be neither tissue- nor development-specific. Non-muscle isoform 2 is the dominant splice variant expressed in various tissues. The Telokin isoform, which binds calmodulin, has been found in a wide variety of adult and fetal tissues. MLCK is probably down-regulated by phosphorylation. The protein contains 1 fibronectin type III domain and 9 immunoglobulin-like C2-type domains.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
kinase-related protein; MLCK; mylk; Myosin light chain kinase; myosin light chain kinase, smooth muscle; myosin, light polypeptide kinase; smooth muscle myosin light chain kinase; telokin
9530072E15Rik; A930019C19Rik; AW489456; BOS_827; KRP; MLCK; MLCK108; MLCK210; MYLK; nmMlck; smMLCK