Sialyltransferase inhibitor 3 fax peracetyl neu5ac

UGCG inhibitors MZ31 (used concentration: 2μM), MZ21 (2μM), miglustat (100μM) were produced as previously described (Ghisaidoobe et al., 2014 (link)) and eliglustat (200nM) was obtained from Bio-Connect. Swainsonine (20μg/mL) and kifunensine (20μM) were obtained from Sigma. N-glycosylation inhibitor activity was confirmed by incubating W6/32 immunoprecipitated HLA-I molecules with Endoglycosidase H (Sigma) in a 20μl reaction mixture (50μM Sodiumcitrate (pH5.5), 0.2% SDS, 2μl Endoglycosidase H and protease inhibitors) for 18h at 37°C followed by HLA-I detection on immunoblot. Sialyltransferase inhibitor (3Fax-peracetyl Neu5Ac, 100μM) was obtained from Sigma and fucosyltransferase inhibitor (2-Deoxy-2-fluoro-L-fucose, 100μM) was obtained from Carbosynth. Cells were cultured for 2 or 3 days at 37°C with inhibitors and analyzed by flow cytometry. Cells were incubated with neuraminidase (N2876, Sigma, 225mU/mL) for 1h at 37°C.