KIF5B-RET phosphorylates and activates STAT3 at different levels in positive cell. A. KIF5B-RET phosphorylates and activates STAT3 both Tyr705 and Ser727 in positive cells. Western blots were performed on 293 T carrying KIF5B-RET or empty vector cells lysates using the indicated antibodies. B. JAK2 and c-Src kinase are involved in KIF5B-RET mediated-STAT3 Tyr705 phosphorylation. 293 T cells carrying KIF5B-RET were pretreated with 15 μM AG490 (Jak2 inhibitor) or 5 μM PP1 (Src inhibitor) for the indicated times before cell lysis. Western blot was then performed using the indicated STAT3 antibodies. C. KIF5B-RET directly interacts STAT3 in positive cells. Enhanced KIF5B-RET expressing 293 T cell lysates were co-immunoprecipitated with control IgG or FLAG antibody. The immunoprecipitates were then subjected to western blot analysis with the indicated antibodies. D. KIF5B-RET directly phosphorylates STAT3 Tyr705. A bacterial-expressed GST- STAT3 fusion protein was incubated in vitro with KIF5B-RET IG that had been immunoprecipitated from 293 T carrying KIF5B-RET cell lysates. Western blots were then performed on the reaction mixtures using the indicated STAT3 antibodies. E. KIF5B-RET also induces a Ras/ERK1/2/STAT3 Ser727 pathway. Enhanced KIF5B-RET expressing 293 T cells were treated overnight with U0126 (MEK inhibitor), and cell lysates were analyzed by western blot using the indicated antibodies. F. Expression of cyclin D1, VEGF, and ICAM-1 in KIF5B-RET positive cells. Two clones of the A549 and BEAS-2B cells, which exhibited stable KIF5B-RET expression, were analyzed cyclin D1, VEGF and ICAM-1 expression by RT-PCR, the products were separated by gel electrophoresis and visualized using ethidium bromide staining.