Hornakova, T., Staerk, J., Royer, Y., Flex, E., Tartaglia, M., Constantinescu, S.N., Knoops, L. and Renauld, J.C.
Notes: The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase® Reporter Assay System. The ProFection® Mammalian Transfection System—Calcium Phosphate was used to transfect 106 HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 106 HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies. (4025)