Fig. 1.

Kidney cell LLOs are hydrolyzed by STT3B-OST. Permeabilized HEK293 cells (WT, STT3A-KO, or STT3B-KO) were incubated with nucleotide sugars and control (CP) or acceptor (AP) tripeptide for 60 min at 37 °C. OST products (glycopeptides and fOSs) from the same dishes were analyzed by FACE and normalized to total cell protein. (A) FACE gels of samples from duplicate dishes. Transferase products were recovered with Endo H; thus positions of LLO glycans are shown with original reducing-end GlcNAc removed. G₃M₉Gn₂ is shown for LLO hydrolysis products, which retain original reducing-end GlcNAc. fOSs in vesicular compartments at the beginning of the incubation are evident (blue bracket). (B and C) Quantitation of OST transferase (B) and hydrolysis (C) reactions (n = 4; see also SI Appendix, Fig. S1). Both were inhibited by 5 μM NGI-1, and AP suppresses hydrolysis. ns, not significant.