Pngase

Transfected HEK‐293 cells were washed with 1× PBS and scraped in RIPA buffer (Tris pH 7.4, NaCl 150 mM, EDTA, 0.5 mM, 1% Triton), containing protease inhibitors (Roche Applied Sciences, Penzberg, Germany). Lysates were cleared by centrifugation at 16,000g at 4°C for 25 min and levels of total cellular protein tested using the DC protein assay kit (BioRad, Hercules, CA, USA). Total protein (25 μg) was treated with PNGase (NEB) following manufacturer's protocol or was left untreated.
PNGase‐treated and ‐untreated protein lysate were separated using 6% Tris‐glycine gels (Invitrogen) and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Blots were then probed with GFP (D5.1) XP Rabbit mAb #2956 (Cell Signaling Technologies, Danvers, MA, USA; 1:1,000) and lamin antibody (Abcam; ab133741, 1:1000). Secondary antibodies were purchased from Dako (Santa Clara, CA, USA) and signal was detected using the chemiluminescent HRP substrate (Millipore). Densitometry was performed with ImageJ software (NIH).

Surface biotinylation was carried out essentially as in Hanus et al. (2016) (link). Dissociated neurons were washed in imaging buffer containing (in mM): 120 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl_2, 15 glucose, 10 HEPES pH 7.4. Cells were then treated with the same buffer containing NHS-SS-biotin (0.8 to 1 mg/mL, Thermo) at room temperature for 7 min. Cells were rinsed and excess biotinylating reagent was quenched using the same buffer supplemented with 10–20 mM L-lysine. Cells were lysed in PBS containing 1% triton X-100, 0.6%SDS and a protease inhibitor cocktail. Biotinylated proteins were purified from cell lysates over streptavidin-conjugated agarose beads and eluted by reduction of disulfide-linked biotin with 50 mM DTT for 15 min at 75 ˚C. Purified surface fractions were divided and either left untreated or treated with endoHf (New England Biolabs) or PNGase (New England Biolabs) according to the manufacturer’s insctructions. Extracts were diluted (~1.5 fold) in sodium phosphate (50 mM, pH 5.5 final) or sodium citrate buffer (50 mM pH 7.5) plus NP40 (or triton X-100, 1% final) for PNGase and endoH respectively. Enzymes were used at 1000 (PNGase) or 3000 (endoH) units/µg total protein and incubated overnight at 37 ˚C.

EndoH and PNGase were used according to the manufacturer’s instructions (New England Biolabs, Hitchin, UK). Murine monoclonal antibodies were used to detect human HLA-I heavy chain (HC10 hybridoma kindly provided by Professor Hidde Ploegh, Boston Children’s Hospital, USA) and the V5-tag (MCA1360, cloneSV5-Pk1, BioRad, Watford, UK) while rabbit polyclonal antibody specific for actin (A2066, Sigma-Aldritch, Gillingham, UK) was used to check sample loading. Western blots were performed as described previously [27 (link)].

Glycoproteins in the cell lysates (20 μg of protein) or medium that were pulled down with heparin-agarose (0.5 ml) were denatured and incubated with Arthrobacter ureafaciens sialidase (Nacalai Tesque, 4 milliunits) and/or O-glycosidase (New England BioLabs, 80,000 units), or peptide N-glycanase (PNGase, New England Biolabs, 1000 units) for 6 h. To remove O-GlcNAc, cell lysates (100 μg of protein) were incubated with O-GlcNAcase (5 μg, R&D Systems) at 37 °C for 2 h.

CHO cells were tranduced with either Ad-tTA (25 MOI) or Ad-197 (20 MOI) and Ad-tTA (5 MOI). At 24 h post transduction (p.t.), the cells were washed with PBS, overlaid with DMEM (Cys⁻/Met⁻), and incubated for 1.5 h. The cells were pulsed with 300 µCi/10⁶ cells for 45 min and the label was chased for the indicated time intervals. CHO cells were washed with ice-cold PBS and lysed with ice-cold PBS-1% NP-40 buffer. Cell lysates were pre-cleared with agarose beads and immunoprecipitated with αFLAG Ab conjugated to agarose beads (Sigma-Aldrich, St. Louis, MO). The samples were eluted from the beads with 50 mM NaOAc-0.15% SDS buffer (10 min, 98°C) and treated with EndoH (Roche Diagnostics, Indianapolis, IN) or PNGase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocols. The samples were separated on a 6% polyacrylamide gel.

Preprocessed cells were washed and lysed in lysis buffer (Beyotime, CHN) containing protease and phosphatase inhibitor cocktails and were further denatured in glycoprotein‐denaturing buffer at 100 °C for 10 min. The cooled lysate was then supplemented with PNGase (NEB, USA) to remove N‐linked glycoproteins. Next, the cell lysate was washed and incubated with succinylated wheat germ agglutinin (sWGA) biotin conjugated beads (Vector Laboratories, USA) at 4 °C overnight. Then, the immunoprecipitated complexes were eluted for further immunoblotting analyses.