Phosphorylated CDCP1 is efficiently pulled down by recombinant SHP2 substrate trapping mutant or SHP2-SH2 domains.

Abstract

<p>HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO₃), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123472#pone.0123472.g004" target="_blank">Fig 4A and 4B</a>, compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.</p