Protein-tyrosine kinase assays were performed in 384-well plates using the Z’-lyte kinase assay system and Tyr2 peptide substrate (Invitrogen) as described elsewhere (19 (link)). Chemical libraries were purchased from ChemDiv, Inc. and included a kinase-directed library (2500 compounds) a phosphatase-directed library (2500 compounds) and a diversity set (5040 compounds). Library screens were conducted in 384-well plates in a final volume of 10 µl per well. Compounds were added to each well (10 µM final), followed by a preformed complex of Hck-YEEI (10 ng/well) and Nef (1:20 molar ratio) plus the substrate peptide (2 µM). Reactions were initiated by the addition of ATP (50 µM final) and incubated at room temperature for 35 min. Reactions were developed and terminated as per the manufacturer’s protocol and fluorescence ratios were calculated as described in the text and elsewhere (19 (link)).