Nef Alleles from All Major HIV-1 Clades Activate Src-Family Kinases and Enhance HIV-1 Replication in an Inhibitor-Sensitive Manner
Figure 5
Primary M-group HIV-1 Nef proteins strongly activate Lyn in cells.
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 [52], 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 (pTyr) 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 [52]. This experiment was repeated three times with comparable results.