Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes

Mahima Swamy, Yogesh Kulathu, Sandra Ernst, Michael Reth, Wolfgang W.A. Schamel (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a ∼400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.

Original languageEnglish
Pages (from-to)131-137
Number of pages7
JournalImmunology Letters
Volume104
Issue number1-2
Early online date1 Dec 2005
DOIs
Publication statusPublished - 15 Apr 2006

Keywords

  • BN-PAGE
  • Drosophila S2 cells
  • Gads
  • SLP-65
  • SLP-76

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