Primary M-group HIV-1 Nef proteins strongly activate Lyn in cells.

Abstract

<p>Yeast cultures were transformed with a galactose-inducible expression plasmid for Lyn-YEEI, which carries a modified C-terminal tail that enables downregulation in the absence of Csk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>, or the empty expression plasmid as a negative control (Con). Where indicated, Lyn-YEEI cells were co-transformed with galactose-inducible vectors for Nef-ELI, Nef-SF2, and ten primary Nef alleles (A1, A2, B, C, F1, F2, G, H, J, and K). Transformed cells were grown in liquid culture in the presence of galactose at 30°C for 18 h. Protein extracts were separated via SDS-PAGE and immunoblotted for tyrosine-phosphorylated proteins (<i>pTyr</i>) as well as for Nef, Lyn and actin as a loading control. Nef-ELI is unable to interact with Lyn and serves as an additional negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032561#pone.0032561-Trible2" target="_blank">[52]</a>. This experiment was repeated three times with comparable results.</p