Western blot analysis of extracts from 293 cells, untreated or treated with hTNF-α #8902 (20 ng/ml for 20 minutes), using Phospho-CYLD (Ser418) Antibody (upper) or CYLD Antibody #4495 (lower).
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 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: 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
Phospho-CYLD (Ser418) Antibody detects endogenous levels of CYLD protein only when phosphorylated on Ser418.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser418 of human CYLD protein. Antibodies are purified by protein A and peptide affinity chromatography.
CYLD is a cytoplasmic deubiquitinating enzyme encoded by a tumor suppressor gene altered in individuals diagnosed with cylindromatosis, a genetic condition characterized by benign tumors of skin appendages (1,2). Functional CYLD deubiquitinase regulates inflammation and cell proliferation by down regulating NF-κB signaling through removal of ubiquitin chains from several NF-κB pathway proteins (3,4). CYLD is a negative regulator of proximal events in Wnt/β-catenin signaling and is a critical regulator of natural killer T cell development (5,6). The transcription factor Snail can inhibit CYLD expression, resulting in melanoma cell proliferation (7).
CYLD can be phosphorylated at Ser418 by the noncanonical IKK family member IKKε. Phosphorylation at Ser418 decreases CYLD deubiquitinase activity and is important for IKKε-driven transformation (8).
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