P 3fax neu5ac

Doxycycline hyclate (2 μg/ml, Acros Organics). EIPA (25–75 μM, MilliporeSigma). Tunicamycin (10 μg/ml, MP Biomedicals). Brefeldin A (10 μg/ml, Cayman Chemical). Monensin (25 nM, Alfa Aesar). P-3FAX-Neu5Ac (Tocris) was used at 100 μM unless stated otherwise. SR-7826 (BioVision) and (–)-Blebbistatin (Biogems) were used at 3.33 μM, unless stated otherwise.

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.