Hek293 cell line

The HEK293 cell line (JCRB9068, Graham, F.L. established) was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). JAK1 complementary DNA carried on the pFN21A vector (Halotag ORF Clone FHC01306) was purchased from the Kazusa DNA Research Institute (Chiba, Japan), and the JAK1 mutation c.1786C>G;p.H596D was introduced to FHC01306 by Promega Japan (Tokyo, Japan). The HEK293 cells were cultured in DMEM containing 1.8 mM calcium supplemented with 10% fetal bovine serum at 37°C with 5% CO₂. For the transfections, the HEK293 cells were cultured in 12-well dishes and then transfected with wild-type JAK1, mutant JAK1 (H596D), or HaloTag Control Vector plasmids using Screen Fect A plus transfection reagent (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) according to the manufacturer’s protocol. Cells were cultured for 24 or 48 hours after being transfected with the indicated plasmids and were collected for Western blotting analysis.

The HEK293 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco/Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, Moregate Biotech, Bulimba, Australia) and 1% antibiotic-antimycotic reagents (Gibco/Life Technologies). The SHSY-5Y and CRL-2061 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SHSY-5Y cells were cultured in a 1:1 mixture of DMEM and Ham’s F12 (Wako Pure Chemical Industries Ltd., Osaka, Japan), and CRL-2061 cells were cultured in RPMI medium (Gibco/Life Technologies). These were supplemented with 10% FBS (Gibco/Life Technologies) and 1% antibiotic-antimycotic reagents. All cell lines were cultured at 37 °C in a 5% CO₂ humidified incubator. Prior to use, cultured cells were rinsed twice with phosphate-buffered saline (PBS, Sigma–Aldrich Co., St. Louis, MO, USA) and collected using a cell scraper.