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. 2015 Oct 28;5:15749. doi: 10.1038/srep15749

Figure 1. Tyrosine phosphorylation-dependent in vitro interaction of CagA with SHP2.

Figure 1

(a) Schematic representation of H. pylori CagA variants used in this work (upper). Strategy for tyrosine phosphorylation of recombinant CagA in E. coli (lower). E. coli BL21(DE3) was co-transformed with a GST-fused CagA expression vector and a v-Src expression vector. (b) Recombinant CagA proteins [CagA(Cn)-His] containing various numbers of EPIYA-C segments (C1, C2, C3, C5, and C8) were purified from E. coli co-expressing v-Src and subjected to immunoblotting with an anti-phosphotyrosine (pY) antibody (left) or CBB staining (right). S: supernatant of E. coli lysates containing GST-fused CagA, P: purified CagA after the cleavage of GST-tag. (c) GST pull-down assay was performed using tyrosine-phosphorylated CagA shown in (b) and GST-fused N-terminal SHP2 fragment containing two tandem-repeated SH2 domains, either functionally active (GST-SHP2/SH2) or dead (GST-SHP2/SH2D). CagA bound to GST-SHP2/SH2 was detected by silver staining. (d) GST pull-down assay was performed using GST-SHP2/SH2 and CagA containing three EPIYA-C segments (C3) or PR-CagA purified from E. coli expressing v-Src (lanes 1–3, 9, 10). GST pull-down assay was also performed using GST-SHP2/SH2 and CagA proteins containing various numbers of the EPIYA-C segments (C1, C2, C3, C5, C8) purified from E. coli without v-Src expression (lanes 4–8, 11–15). CagA bound to GST-SHP2/SH2 was detected by silver staining.