Supplemental Experimental Procedures

calphostin C, latrunculin B, jasplakinolide, and cytochalasin B (Calbiochem). Anti-MHCI (W6/32) was either purified from culture supernatants, or used as W6/32-biotin conjugates (Serotec). Purified anti-CD80 and CD86 mAbs (eBiosciences) were labeled by either Cy5 (Amersham) or with Alexa dyes (Molecular Probes). Antibodies against Src and pTyr418-Src, pTyr, TIAM, Hck and Fyn (Cell Signaling Technology, Calbiochem and Upstate), Rac (BD Transduction Labs), PAK-PBD-coupled-agarose (Chemicon) were also used. Labeled secondary detection reagents used included goat anti-mouse IgG (Fc)-PE, donkey anti-rat IgG (Fc)-PE (Jackson ImmunoResearch), streptavidin-PE, streptavidin-CyChrome, and streptavidin-PE-TxR (BD PharMingen). For studies with HAp-tagged molecules, intracellular HAp staining was done using an anti-HAp mAb (Cell Signaling Technology). Similarly, gene products with the myc-p-tag were detected in permeabilized cells with an anti-myc-p mAb (Cell Signaling Technology). The CA, KD and DN src gene constructs were made by cloning PCR-amplified versions of appropriate cDNAs into myc-p containing pCDNA3.1 vector. Wild type (WT), constitutively active (CA; Y529F), dominant negative (DN; K296R, Y528F), and kinase dead (KD; K279R) isoforms of human Src obtained from Upstate Cell Signaling Solutions (USA) were cloned into a mammalian expression vector, pCDNA3.1(B-)MycHis+ using availabl