The Arp2/3 complex is threonine- and tyrosine-phosphorylated in cells. (A) The Arp2/3 complex purified from A. castellani separated by 2D electrophoresis revealed that Arp2 migrated as five spots and Arp3 as four spots (arrowheads). Pretreatment of the complex with the dual-specificity AP resulted in Arp2 migrating as two spots and Arp3 migrating as two spots at a more basic pI relative to a fiduciary marker (asterisks). (B) The Arp2, Arp3, and ARPC1 subunits of the Arp2/3 complex purified from A. castellani labeled with antibodies to pThr and pTyr. Dephosphorylation of the Arp2/3 complex with AP abolished labeling of Arp2 with pThr and pTyr antibodies, decreased labeling of ARPC1 by pTyr antibodies to ARPC1, and did not change labeling of Arp3. Dephosphorylation with the tyrosine-specific phosphatase YOP did not affect labeling of pThr antibodies but abolished labeling of Arp2 with pTyr antibodies and decreased labeling of ARPC1. (C) The Arp2 subunit of purified bovine Arp2/3 complex labeled with antibodies to both pThr and pTyr. (D) The Arp3 and Arp2 subunits of recombinant human Arp2/3 complex labeled with antibodies to pThr and pTyr.