Fig. 3. G5-7 directly binds and inhibits JAK2 kinase activity.

(A) Proliferation of U87MG/EGFRvIII cells treated with G5-7, -8, or -9, orbiotin-labeled G5-7 and G5-8 (compounds “D” and “E,” respectively, right). Data are means ± SEM from three experiments. (B) Biotin-streptavid in pull-down assay with NeutrAvidin beads was performed in cells treated with biotinylated G5-7 (“D”) or G5-8 (“E”) alone or in combination with G5-7, analyzed by silver staining (lower panel), and immunoblotted for JAK2 (upper panel). (C) The interaction between JAK2 and G5-7 (left blots) or biotinylated G5-7 (“D”; right blots) was mapped by immunoprecipitation (IP) using full-length JAK2 (FL) or fragments of JAK2: ATD, N-terminal domain; CTD, C-terminal domain; PKD, pseudokinase domain; and fragments 1 to 5 with corresponding AA region. (D) Effect of G5-7 on EGFR phosphorylation at Tyr¹⁰⁴⁵ and Tyr¹⁰⁶⁸. Labels between the graphs and blots apply to both charts and blots. Blots are representative and data are means ± SEM of three experiments. *P < 0.01, **P < 0.01, two-tailed Student’s t test. (E) Analysis of the in vitro kinase activity of JAK2, immunoprecipitated from human embryonic kidney (HEK) 293 cells and preincubated with G5-7, G5-9, or lestaurtinib, on kinase-deficient EGFR (top) or STAT3 (bottom). Data are means ± SEM (*P< 0.05, one-way ANOVA, n = 3). (F) Effect of G5-7 on the interaction between the N-terminal fragment of JAK2 [construct #2 in (C)] and EGFR. Interaction between full-length JAK2 (#1) and EGFR serves as control. Blots are representative of three experiments.