“The deglycosylation experiments with PNGase F (Sigma) were carried out according to the manufacturer's instructions.”
“The purified RNase B (Sigma) was used as a positive control in parallel in the N-deglycosylation experiment and the purified bovine fetuin (Sigma) as a positive control in the O-deglycosylation experiment.”
“Cross-contamination of cytosolic proteins into the membrane fraction is typically Total protein (50-100 µg) from RGC5 cells or those transfected with pTarget-FLAG-OPTN was digested with PGNase F (0.25 units, Enzymatic In-Solution N-deglycosylation kit, Sigma) for N-deglycosylation or with O-glycosidase (2.5 mUnits, Sigma) without or with addition of sialidase A (5 mUnits, Sigma) for O-deglycosylation per manufacturer's instructions.”
“Lysates from RGC5 cells were treated with peptide N-glycosidase F (PNGase F, for N-deglycosylation) or O-glycosidase (for O-deglycosylaton) without or with α-2 (3,6,8,9) - neuraminidase (sialidase), and Western blotted for optineurin.”
“Lysates from rat retinal ganglion RGC5 cells were subjected to N - or O-deglycosylation or membrane protein extraction.”
“The plant cell system used for the expression of prGCD has the potential to be highly advantageous due to the safe and cost effective production of therapeutic recombinant glycoproteins that are not suitable for production in bacterial hosts, and are currently being expressed in costly mammalian expression systems. prGCD is produced in carrot cells, naturally possesses terminal Mannose residues and therefore, prGCD production does not require costly enzymatic deglycosylation as would be required for the production of recombinant GCD in mammalian cells”
“A) or O-glycosidase (for O-deglycosylation) without (B”
“Lysates from RGC5 cells or those transfected with pOPTN-FLAG were untreated (control), or treated with PNGase F (for N-deglycosylation,”
“By Coomassie blue staining, the purified RNase B, as a positive control, had a band shift from 17 to 15 kDa after N-deglycosylation (”
“In parallel, the positive controls, ribonuclease B (RNase B) and fetuin, showed a band shift after N - or O-deglycosylation respectively (”
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