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. 2002 Jun;22(12):4241–4255. doi: 10.1128/MCB.22.12.4241-4255.2002

FIG. 9.

FIG. 9.

SLAP-2 expression promotes degradation of c-Cbl substrates SYK and ZAP-70. Mammalian expression constructs for SYK (a) or ZAP-70 (b) were cotransfected into COS-7 cells with either empty vector or expression constructs encoding MYC-tagged wild-type (WT) SLAP-2 and SH2 mutant (SH2*) SLAP-2. Lysates from transfected cells were subjected to SDS-PAGE and immunoblotted with either anti-SYK or anti-ZAP-70 (top panels), anti-MYC (middle panels), and anti-β-tubulin (bottom panels) antibodies. UT, untransfected. (c) Transfected SLAP-2 binds to endogenous c-Cbl in COS cells. A lysate from COS-7 cells transfected with MYC-tagged wild-type SLAP-2 was immunoprecipitated with anti-c-Cbl antibody. Membranes were immunoblotted with anti-c-Cbl (top panel) and anti-MYC (bottom panel) antibodies. IP, immunoprecipitation; IgH, immunoglobulin heavy chain. (d) Jurkat p116 T cells were either left untransfected (UT) or transfected with empty vector pEF or wild-type (WT) SLAP-2, ΔC mutant SLAP-2, or ZAP-70 expression constructs. Cells were either left unstimulated (−) or stimulated with anti-CD3 antibody (+). A 40-μg portion of whole-cell lysate was subjected to SDS-PAGE and immunoblotted with anti-ZAP-70 (top panel), anti-MYC (middle panel), and anti-β-tubulin (bottom panel) antibodies. (e) A 500-μg portion of lysate from the experiment described in panel d was immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with anti-SLP-76 antibody.