Figure 2

Hck activation. (A) HEK293 cells were transfected with cDNA encoding GFP alone or Nef-GFP fusion proteins in the presence of Hck plasmid, and analyzed by Western blotting with anti-GFP (FL; Santa Cruz), anti-Hck (clone 18; Transduction Laboratories, Lexington, KY), anti-Hck phosphorylated at tyrosine 411 (Hck-pTyr411; Santa Cruz) and anti-phosphotyrosine (PY99; Santa Cruz) as indicated. The Nef variants tested are indicated in the figure. A representative datum set of 3 independent experiments is shown. (B) Quantification of Hck activation by Nef. The indicated values represent the Hck activation activity after the level of phosphorylated Hck had been normalized to the amounts of total Hck. The values presented were calculated from the data shown in panel A, and are relative to the wild-type control arbitrarily set to 100%. Data represent the means ± SD of 3 independent experiments, and statistical analysis was performed based on ANOVA with multiple comparisons vs. wt (Bonferroni t-test). n.s., not significant.