<p>A) SDS-PAGE of recombinant purified proteins. Each of the indicated Nef subtypes were expressed in bacteria and purified with N-terminal His-tags (<i>left</i>). GST and GST fusion proteins with the Hck SH3 domain (wild-type and W93A mutant) were also expressed in bacteria and immobilized on glutathione-agarose beads (<i>right</i>). Protein aliquots were resolved by SDS-PAGE and stained with Coomassie blue. B) Nef-SH3 binding assay. Recombinant His-tagged Nef proteins (1 µg) were incubated with equimolar amounts of immobilized GST and GST-SH3 fusion proteins. The agarose beads were then washed, and associated Nef proteins were detected by immunoblotting using antibodies to the His-tags. C) Nef-induced Hck activation. Kinase activity of recombinant downregulated Hck (Hck-YEEI; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-EmertSedlak1" target="_blank">[53]</a> was determined in the absence or presence of the indicated Nef proteins using the Z-Lyte assay as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#s3" target="_blank">Materials and Methods</a>. Results are expressed as the mean percent of maximum substrate phosphorylation ± S.D.; this experiment was repeated twice with comparable results.</p
