Ac4mannaz

Sodium chloride, magnesium chloride, calcium chloride, ethylenediaminetetraacetic acid disodium salt dihydrate, 2-mercaptoethanol, glycerol, NGI-1, kifunensine, swainsonine, chloroform, dibenzocyclooctyne-PEG4-biotin (DBCO-biotin) and Tween-20 were purchased from Sigma-Aldrich. Acid Blue 9 (AB9) and a Minute plasma membrane-derived lipid raft isolation kit were purchased from Fisher Scientific. Ac₄ManNAz was purchased from Click Chemistry Tools. Neu5Ac was purchased from Cayman Chemical. Bovine serum albumin (BSA), phi29 DNA polymerase, dNTP, T4 DNA ligase, ATP, proteinase K, α2-3,6,8,9-neuraminidase A, PNGase-F and O-glycosidase were purchased from New England BioLabs (NEB). TRIzol, RNase A, RNase T1, formamide, HBSS and CellTracker Orange CMRA were purchased from Thermo Fisher Scientific. RNA Clean and Concentrator 5 kits were purchased from Zymo Research. Intercept (PBS) blocking buffer and IRDye 800CW streptavidin were purchased from Li-Cor Biosciences. The 0.45-μm nitrocellulose (NC) membrane was purchased from Cytiva Life Sciences. All the oligonucleotide sequences were purchased from Integrated DNA Technologies and were purified by high-performance liquid chromatography or polyacrylamide gel electrophoresis and confirmed by mass spectrometry (Supplementary Table 1). All other reagents and solvents were obtained from the domestic suppliers and were used as received.

Stocks of azide-labeled sugars N-Acetyl-9-azido-9-deoxy-neuraminic acid (9Az sialic acid, Carbosynth) and N-azidoacetylmannosamine-tetraacylated (Ac₄ManNAz, Click Chemistry Tools) were made to 500 mM in sterile dimethyl sulfoxide (DMSO). Stocks of unlabeled sugars N-Acetyl-D-galactosamine (GalNAc, Sigma) and D-(+)-Galactose (Gal, Sigma) were made to 500 mM and 50 mM, respectively, in sterile water. In cell experiments ManNAz was used at a final concentration of 100 μM. In vitro experiments with ManNAz used 0, 2, or 20 mM ManNAz (up to 200× the in-cell concentrations) for 2 h at 37°C. The in-cell experiments with 9Az sialic acid used a 1.75 mM final concentration for between 6 and 48 h. Gal and GalNAc were used as media supplements at 10 μM and 100 μM, respectively, and were added simultaneously with ManNAz for labeling.
Working stocks of glycan-biosynthesis inhibitors were all made in DMSO at the following concentrations and stored at −80°C: 10 mM NGI-1 (Sigma), 10 mM Kifunensine (Kif, Sigma), 10 mM Swainsonine (Swain, Sigma), 50 mM P-3F_AX-Neu5Ac (Tocris). All compounds were used on cells for 24 h and added simultaneously with ManNAz for labeling.