(A) Schematic representation of paxillin with selected binding partners (bold lines) and upstream kinases (arrows) to the corresponding phosphorylated residue. (B) In vitro binding assay of semisynthetic paxillin (4a) with a negative control (GST), and GST-fusions of the paxillin binding regions of FAK, GIT, and PTP-PEST. The GST and GST fusions were bound to solid support, incubated with 4a, washed, and then probed for paxillin binding with an anti-hexahistidine Western blot. (C) Phosphorylation of semisynthetic Y31Pax (4a). Paxillin was treated with Src, ERK, JNK, or no kinase in the presence of MgCl2 and ATP, and phosphorylation was visualized by phosphospecific anti-paxillin [pY31], [pY118], [pS126], and [pS128]. The reactions were also probed with a general anti-paxillin (pan) antibody to demonstrate comparable loading. (−) Negative control treated with no kinase.