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Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 10 min at room temperature. A suggested positive control is placenta tissue.
The eight members of the recently identified Suppressor of Cytokines Signaling (SOCS) family are SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, and CIS. Structurally the SOCS proteins are composed of an N- terminal region of variable length and amino acid composition, a central SH2 domain, and a C-terminal motif called the SOCS box. The SOCS proteins appear to form part of a classical negative feedback loop that regulates cytokine signal transduction. Transcription of each of the SOCS genes occurs rapidly in vitro and in vivo in response to cytokines, and once produced, the various members of the SOCS family appear to inhibit signaling in different ways. SOCS1 and SOCS6 interact with the insulin receptor (IR) when expressed in human hepatoma cells (HepG2) or in rat hepatoma cells overexpressing human IR. SOCS1 and SOCS6 inhibit insulin-dependent activation of ERK1/2 and protein kinase B in vivo and IR- directed phosphorylation of IRS1 in vitro. These results suggest that SOCS proteins may be inhibitors of IR signalling and could mediate cytokine-induced insulin resistance and contribute to the pathogenesis of type II diabetes.
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