Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight), and then either untreated (-) or transfected with poly(dA:dT) (0.004 U/ml; 7 hr; +), using Phospho-TRAF2 (Ser11) (E2B6L) Rabbit mAb (upper) or TRAF2 (C192) Antibody #4724 (lower).
Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight), untreated or LPS-treated (1 μg/ml for indicated times), using Phospho-TRAF2 (Ser11) (E2B6L) Rabbit mAb (upper) or TRAF2 (C192) Antibody #4724 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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-TRAF2 (Ser11) (E2B6L) Rabbit mAb recognizes endogenous levels of TRAF2 protein only when phosphorylated at Ser11.
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser11 of human TRAF2 protein.
TRAFs (TNF receptor-associated factors) are a family of multifunctional adaptor proteins that bind to surface receptors and recruit additional proteins to form multiprotein signaling complexes capable of promoting cellular responses (1-3). Members of the TRAF family share a common carboxy-terminal "TRAF domain", which mediates interactions with associated proteins; many also contain amino-terminal Zinc/RING finger motifs. The first TRAFs identified, TRAF1 and TRAF2, were found by virtue of their interactions with the cytoplasmic domain of TNF-receptor 2 (TNFRII) (4). The six known TRAFs (TRAF1-6) act as adaptor proteins for a wide range of cell surface receptors and participate in the regulation of cell survival, proliferation, differentiation, and stress responses.
TRAF2 is phosphorylated at Ser11 by IKK-ε, promoting K63-linked ubiquitination, NF-κB activation, and cellular transformation (5).
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