Peptide n glycosidase f pngase f

Endoglycosidase H (Endo H) and peptide: N-glycosidase F (PNGase F) were purchased from New England Biolabs (Beverly, MA). Deglycosylation assays were performed according to the manufacturer's instructions.

C57BL/6 mice of 8 weeks years old (n = 120) were purchased from Shanghai jiesijie experimental animal Co., Ltd. All animal experiments were in accordance with the requirements of the Animal Experiment Ethics Committee of Fudan University and were approved. Animals were maintained on commercial normal diet (proteins 24.02%, fat 12.95%, carbohydrates 63.03%, 3.44 kcal/g) from Beijing Keao Xieli Feed Co., Ltd. Complete diet composition is listed in Table S1. The environment was kept at 23 ± 2°C, and 12 h light/dark cycle with light on at 06:00 a.m.
Reagents: Sodium dodecyl sulfate (SDS), 1‐hydroxybenzotriazole monohydrate (HOBt), sodium borodeuteride (NaBD₄), formic acid (FA), trifluoroacetic acid (TFA), sodium hydroxide (NaOH), super‐2,5‐dihydroxybenzoic acid (super‐DHB), 10 × phosphate buffered solution (PBS), dimethyl sulfoxide (DMSO) and Sepharose CL‐4B were purchased from Sigma Aldrich. Acetonitrile (ACN) and ethanol (EtOH) were provided by Merck Millipore. NP‐40 and peptide‐N‐glycosidase F (PNGase F) were from New England Biolabs (Inc.). The FiltrEX™ 96‐well Clear Filter Plates with 0.2 μm PVDF Membrane were from Corning. Milli‐Q water (MQ) was provided by Milli‐Q system.

DMEM, FBS, penicillin-streptomycin, trypsin-EDTA solution, Dil-labeled human LDL, and unlabeled human LDL were obtained from ThermoFisher Scientific. Complete EDTA-free protease inhibitors and X-tremeGENE^TM HP DNA transfection reagent were purchased from Millipore Sigma. Peptide-N-glycosidase F (PNGase F) was obtained from New England Biolabs (Beverly, MA). All other reagents were obtained from Fisher Scientific, unless otherwise indicated.

Peptide-N-glycosidase F (PNGase F) (catalogue number P0705S), O-glycosidase (catalogue number P0733S), and α2-3,6,8,9-neuraminidase A (catalogue number P0722S) were purchased from New England BioLabs (NEB) (Ipswich, MA). Eighteen micrograms of 2% n-dodecyl-β-d-maltoside (DMT)-solubilized HeLa S3 membrane proteins (1.4 mg/ml) was first incubated with 10× denaturing buffer in a volume of 20 μl at 100°C for 10 min. The reaction mixture was then incubated with GlycoBuffer 2 and N-glycosidase F (1,000 U); O-glycosidase (80,000 U); both N-glycosidase F (1,000 U) and O-glycosidase (80,000 U); or N-glycosidase F (1,000 U), O-glycosidase (80,000 U), and α2-3,6,8,9-neuraminidase A (80 U) at 37°C for 1 h in a volume of 40 μl. Mock digestion reaction mixtures were set up without the addition of any enzymes.

20 µg of iRhom2 KO and WT THP-1 cell lysate, or iRhom2 KO and WT HTB94 lysate, were treated with endoglycosidase H (Endo H, New England Biolabs, P0702) or Peptide-N-Glycosidase F (PNGase F, New England Biolabs, P0704) according to the manufacturer’s protocol. Afterwards, the samples were separated SDS–PAGE electrophoresis and analysed by Western blotting.