Hek293

Human biliary cancer cells (NOZ), mouse embryonic fibroblasts (MEF) (Japanese Collection of Research Bioresources, Tokyo, Japan), human breast cancer cells (MCF7), human hepatocyte carcinoma cells (HepG2) and human embryonic kidney 293 cells (HEK293) (Riken Cell Bank, Ibaraki, Japan) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with L-glutamine and phenol red (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) in a humidified atmosphere with 5% CO₂ at 37°C. The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin-amphotericin B (Fujifilm Wako).

HEK293 (purchased from ATCC), HEK293/FUT8KO, COS7 (purchased from RIKEN cell bank), and HeLa (purchased from RIKEN cell bank) cells were grown at 37 °C under 5% CO₂ conditions in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 50 μg/ml kanamycin. Dr Jianguo Gu (Tohoku Medical and Pharmaceutical University) kindly provided the HEK293/FUT8KO cells, which were generated by Zn-Finger technology. To investigate FUT8 degradation, MG132 (10 μM final concentration) or CQ (50 μM final concentration) was added to the culture medium 48 h after transfection. After 48 h, the cells were collected for Western blotting.

HEK293 and COS-7 cells were purchased from RIKEN Cell Bank (Japan, HEK293: RCB1637, COS-7: RCB0539). For transient transfection of expression plasmids, 2x10⁵ HEK293 or COS-7 cells were seeded on 35 mm dishes and incubated overnight at 37°C with 5% CO2. The appropriate combinations of expression plasmids (total 1 μg/dish) were transfected into the cells using the X-tremeGENE 9 DNA transfection reagent (Sigma). The culture medium was changed 24-hours later. After an additional 24-hour incubation, the cells were washed twice with ice-cold PBS and harvested with Lysis buffer on ice. The cell lysates were centrifuged at 20,000 x g for 15 min at 4°C, and the supernatants were used for several assays.