Inducible activation of NF-κB is inhibited by the atypical SH3-binding motif in SLP-65 and the C-terminal SH3 domain of Grb2. (A) SLP-65–regulated activation of NF-κB. SLP-65–deficient DT40 cells (left bars) and the 2 transfectants expressing either full-length or s-SLP-65 (middle and right bars, respectively) were transfected with an NF-κB reporter construct together with the β-galactosidase expression plasmid. Resulting enzymatic activities of resting (□) and BCR-stimulated cells (▪) were determined as described in Figure 4A. Error bars indicate SD of 6 independent measurements. (B) Impact of the C-terminal Grb2 SH3 domain on NF-κB activation. DT40 cells expressing equal amounts of either wild-type or W193K mutant Grb2 (left and right bars, respectively; also Figure 1D) were transfected with the NF-κB reporter and β-galactosidase constructs, and resulting enzymatic activities in resting (□) and BCR-stimulated cells (▪) were determined as described in Figure 4A. Error bars indicate SD of 3 independent measurements.