Detection of ZAP-70 protein expression and EGCG binding with ZAP-70 and
ZAP-70 deletion mutants. A, expression of ZAP-70 in Jurkat, P116
(ZAP-70-deficient), and P116.cl39 (ZAP-70-restored) cells. ZAP-70 was detected
using specific antibodies as described under “Experimental
Procedures.” Equal protein loading and protein transfer were confirmed
with anti-β-actin. B, ZAP-70-EGCG binding ex vivo.
EGCG-Sepharose 4B affinity chromatography was used to pull down ZAP-70 in
lysates prepared from Jurkat, P116, or P116.cl39 cells. IB,
immunoblot. C, specific binding assay for ZAP-70 and EGCG. The
Kd (dissociation kinetic) value of the EGCG-ZAP-70
interaction (Kd = 0.6207 μm) was obtained by
using a GST-ZAP-70 affinity-binding assay as described under
“Experimental Procedures.” D, schematics of ZAP-70
full-length (ZAP-70 FL) and three deletion mutants (ZAP-70 D1, D2, and D3). A
series of full-length and deletion mutants of ZAP-70 nucleotide constructs was
created as indicated. E, in vitro identification of the EGCG-binding
site of ZAP-70. The full-length and deletion mutants of ZAP-70 were translated
in vivo with l-[35S]methionine using
TnT and subjected to the EGCG-Sepharose 4B pulldown assay.