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For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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: 10
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues at the carboxyl terminus of human DAPK3/ZIPK. Antibodies were purified by protein A and peptide affinity chromatography.
Death-associated protein kinase (DAPK1) is a Ca2+/calmodulin-regulated serine/threonine kinase that participates in a wide range of apoptotic signals including interferon-γ, tumor necrosis factor α, Fas, activated c-Myc, and detachment from the extracellular matrix. In addition to the kinase domain and calmodulin regulatory segment, DAPK1 also has eight ankyrin repeats, a cytoskeleton binding region, and a conserved death domain (1-3). Deletion of the calmodulin-regulatory domain generates a constitutively active mutant kinase. Ectopic expression of wild-type DAPK1 induced cell death in HeLa cells. Conversely, expression of a catalytically inactive mutant protected cells from interferon-γ-induced cell death (4). The catalytic domain of DAPK1 has very high sequence similarity to vertebrate myosin light chain kinase (MLCK) and a RXX(S/T)X motif derived from myosin light chain protein was shown to be phosphorylated in vitro by DAPK1 (5).
The DAPK family consists of several kinases including DAPK, DAPK2/DRP-1 (6), and DAPK3/ZIPK/DLK (7-9) with homology in their catalytic domain. Overexpression of DAPK3/ZIPK, but not a catalytically inactive mutant, can induce apoptosis (7). DAPK3 was also identified as a myosin light chain kinase, demonstrating ability to phosphorylate the regulatory light chain of myosin II in a Ca2+/calmodulin-independent manner (8). In addition to an amino-terminal kinase domain, DAPK3 contains a carboxy-terminal leucine zipper domain that mediates interaction with leucine zipper transcription factors such as ATF4 (7). DAPK3 is predominantly localized to the nucleus and has been found in PML oncogenic domains (PODs) associated with DAXX and PAR-4, and can phosphorylate PAR-4 in vitro (10,11). In addition, DAPK3 can phosphorylate STAT3 at Ser727 to enhance its transcriptional activity (12).
UniProt ID: O43293
Entrez-Gene Id: 1613
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