|
Figure 4.
Figure 4. The Gads SH3 Domain Confers SLP-76 Binding and
TCR Signaling Properties to Grb2(A) Schematic of the Grb2-Gads
chimera that was generated by PCR amplification and ligation of
cDNA fragments encoding amino acids 1–154 of Grb2 (N-SH3 and
SH2) and amino acids 257–322 of Gads (C-SH3). The chimeric
cDNA was cloned into a pEF vector with a Flag epitope tag.(B)
Jurkat T cells were electroporated with 40 μg of either empty
pEF vector, or pEF containing full-length Gads, Grb2, or the
chimera. Anti-Flag immunoprecipitations were performed on
clarified lysates, and Western blots were probed with polyclonal
anti-SLP-76 anti-sera (a gift from Gary Koretzky) (top). Total
cell lysates from each condition were Western blotted with
anti-Flag antibody (Sigma) to assess expression levels of the
transfected proteins.(C) Grb2-Gads chimera synergizes with
SLP-76 to activate NFAT downstream of the TCR. Jurkat cells (2
× 10^7) were electroporated with 20 μg NFAT-luciferase
reporter construct, together with 40 μg of empty pEF vector or
40 μg of Flag epitope-tagged Gads, Grb2, or a Grb2 construct
with the C-terminal SH3 of Gads. Luciferase assays were
performed and data presented as described for Figure 2C.
|
