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. 2000 Jun 13;97(13):7470–7475. doi: 10.1073/pnas.130193097

Figure 5.

Figure 5

SH2D1A activates NF-κB. (A) Wild-type SH2D1A activates NF-κB. An NF-κB-dependent luciferase reporter plasmid with three upstream NF-κB binding sites was transfected into 293T cells with a control β-galactosidase expression construct (12) and pcDNA3 or increasing amounts of pcDNA3-based expression plasmid for FLAG-SH2D1A (F-SH2). Luciferase activities were normalized for cotransfected β-galactosidase activity and pcDNA3 vector control/luciferase reporter activity. Mean relative luciferase activities (±SD) from at least four different experiments are shown. (B) A dominant-negative IKKβ mutant inhibits SH2D1A-induced NF-κB activation. Cells were transfected with 5 μg of pcDNA3-based expression vector for FLAG-SH2D1A (F-SH2) alone or in combination with 1 μg of pcDNA3-IKKβΔ34. Luciferase reporter assays are as in A. (C). SH2D1A mutants also activate NF-κB. Ten micrograms of pcDNA3-based expression vector for FLAG-SH2D1A (WT), FLAG-SH2D1A-R32T (T), FLAG-SH2D1A-P101L (L), or FLAG-SH2D1A-C12 (C12) were transfected into 293T cells. Western blots for SH2D1A protein expression using the M5-FLAG antibody are shown above the respective experiments in A and B.