Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with H2O2 (11 mM, 1 min; +), using Phospho-SLP-76 (Ser376) (D9D6E) Rabbit mAb (upper) or SLP-76 Antibody #4958 (lower).
Western blot analysis of extracts from EL4 cells, untreated (-) or treated with H2O2 (11 mM, 1 min; +), using Phospho-SLP-76 (Ser376) (D9D6E) Rabbit mAb (upper) or SLP-76 Antibody #4958 (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-SLP-76 (Ser376) (D9D6E) Rabbit mAb recognizes endogenous levels of SLP-76 protein only when phosphorylated at Ser376.
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser376 of human SLP-76 protein.
SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is a hematopoietic adaptor protein that is important in multiple biochemical signaling pathways and necessary for T cell development and activation (1). ZAP-70 phosphorylates SLP-76 and LAT as a result of TCR ligation. SLP-76 has amino-terminal tyrosine residues followed by a proline rich domain and a carboxy-terminal SH2 domain. Phosphorylation of Tyr113 and Tyr128 result in recruitment of the GEF Vav and the adaptor protein Nck (2). TCR ligation also leads to phosphorylation of Tyr145, which mediates an association between SLP-76 and Itk, which is accomplished in part via the proline rich domain of SLP-76 and the SH3 domain of Itk (3). Furthermore, the proline rich domain of SLP-76 binds to the SH3 domains of Grb2-like adaptor Gads (3,4). In resting cells, SLP-76 is predominantly in the cytosol. Upon TCR ligation, SLP-76 translocates to the plasma membrane and promotes the assembly of a multi-protein signaling complex that includes Vav, Nck, Itk, and PLCγ1 (1). The expression of SLP-76 is tightly regulated; the protein is detected at very early stages of thymocyte development, increases as thymocyte maturation progresses, and is reduced as cells mature to CD4+ CD8+ double-positive thymocytes (5).
Following TCR ligation, SLP-76 is phosphorylated at Ser376 by the hematopoietic progenitor kinase 1 (HPK1) (6,7). This phosphorylation induces interaction with 14-3-3ε, which leads to the disassembly of TCR signaling complexes and down regulation of TCR signaling (6-8).
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