Tunicamycin

Tunicamycin, 4-phenylbutylate, cycloheximide, ibuprofen, and lysozyme were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The ALDH2 protein (18–517 aa) was obtained from ATGen (Seoul, Korea). (±)-Flurbiprofen were from Sigma (St Louis, MO, USA) or Cayman Chemical (Ann Arbor, MI, USA). α-Lactalbumin, aspirin, and meloxicam were from Sigma. Human recombinant leptin for use in vitro was from Sigma and mouse recombinant leptin for use in vivo was from R'D Systems (Minneapolis, MN, USA). Ferriteglycidyl methacrylate (FG) beads (epoxy beads: TAS8848N1110) were from Tamagawa Seiki (Tokyo, Japan). 4′-hydroxy flurbiprofen was obtained from Toronto Research Chemicals (Toronto, ON, Canada).

The standard technique for the yeast culture and manipulation was used (Guthrie and Fink, 2002 ). Synthetic medium (SC) was prepared as described by Hanscho et al., 2012 (link). 2-Deoxy-D-glucose (2DG), tunicamycin, and MG132 were purchased from FUJIFILM Wako (cat. # 046-06483, 202-08241, and 139-18451, respectively). tunicamycin and MG132 were dissolved in DMSO to make stock solutions (5 mg/ml and 42 mM, respectively). Cells were grown to the mid-log phase at 30°C in SC before imaging unless otherwise noted.

Approximately 2 × 10⁶ of CHO-PIGS KO, HEK293-PIGS KO, PIGS-UBE2J1 DKO and PIGS-SYVN1 DKO cells were precultured in normal medium overnight, cells were washed with wash medium (glucose-free DMEM buffered with 20 mM HEPES) and incubated for 1 h at 37 °C in 1 mL of reaction medium (glucose-free DMEM buffered with 20 mM HEPES, pH 7.4, and supplemented with 10% dialyzed FBS (Gibco), 10 μg/mL tunicamycin (Wako), and 100 μg/mL glucose). [2-³H] Mannose (American Radiolabeled Chemicals) was added to 25 Ci/ml, and the cells were incubated for 1 h at 37 °C in 5% CO₂. After labeling, the cells were washed twice with 1 ml of PBS and released from culture plates by cell scraper. Cells were pelleted and washed with 1 ml of cold PBS. Radiolabeled GPIs were extracted by 1-butanol and separated by HPTLC (Merck)^{74 (link)}, and visualized by a FLA 7000 analyzer (Fujifilm). Quantitative analysis was performed by JustTLC (SWEDAY).

Tunicamycin was obtained from Wako Pure Chemical Industries, Ltd. (Japan). ExoQuick-TC was obtained from System Biosciences (CA). Exosome-depleted FCS was obtained from System Biosciences (CA) or Life Technologies (CA). We wear gloves and lab coat and used hoods for the preparation of biohazard elements.

Tunicamycin was purchased from Wako Pure Chemical Industries (Osaka, Japan). 2-Deoxy-D-glucose and rabbit polyclonal antibodies against glial fibrillary acidic protein (GFAP), doublecortin (DCX), and HRD1 (C-terminal) were purchased from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal antibodies against nestin, βIII-tubulin, neuronal nuclei protein (NeuN; A60), and microtubule-associated protein-2 (MAP-2) were purchased from Millipore (Temecula, CA). A mouse monoclonal antibody against KDEL (10C3) was purchased from Stressgen Biotechnologies (Victoria, British Columbia, Canada). A rabbit polyclonal antibody against cleaved Notch1 (Val1744) was purchased from Cell Signaling Technology (Danvers, MA). A goat polyclonal antibody against Notch3 (M-20) and a rabbit polyclonal antibody against GADD 153 (CHOP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from GE Healthcare (Buckinghamshire, United Kingdom). An Alexa Fluor 488-conjugated anti-mouse IgG antibody and an Alexa Fluor 546-conjugated anti-rabbit IgG antibody were purchased from Life Technologies (Grand Island, NY). Western Lightning Chemiluminescence Reagent Plus was obtained from PerkinElmer Life Science (Waltham, MA).