Endoglycosidase hf

All samples were suspended in SDS sample buffer, treated with 1000 U of Endoglycosidase Hf or Endo H (respectively, for molecular weight proteins ~10-50 kDa or ~50–100 kDa; New England Biolabs, P0703S or P0702S) where indicated, and denatured for 1 h at 37 °C prior to resolution by SDS-PAGE (16% PAGE, 120 V, 120 min). To analyse the translation products, gels were fixed for 5 min (20% (v/v) MeOH, 10% (v/v) AcOH), dried for 2 h at 65 °C and the radiolabelled species visualised using a Typhoon FLA-700 (GE Healthcare) following exposure to a phosphorimaging plate for 24–72 h. Knock-down efficiencies (EMC2, EMC5, Sec61α, SRα) and controls (EMC6, OST, LMNB1, hSnd2, CAML) were determined by quantitative immunoblotting. Following transfer to a PVDF membrane in transfer buffer (0.06 M Tris, 0.60 M glycine, 20% MeOH) at 300 mA for 2.5 h, PVDF membranes were incubated in 1X Casein blocking buffer (10X stock from Sigma-Aldrich, B6429) made up in TBS, incubated with appropriate primary antibodies (1:500 or 1:1000 dilution) and processed for fluorescence-based detection as described by LI-COR Biosciences using appropriate secondary antibodies (IRDye 680RD Donkey anti-Goat, IRDye 680RD Donkey anti-Rabbit, IRDye 800CW Donkey anti-Guinea pig, IRDye 800CW Donkey anti-Mouse) at 1:10,000 dilution. Signals were visualised using an Odyssey CLx Imaging System (LI-COR Biosciences).

Cells were starved in methionine- and cysteine-free DMEM for 30 min at 37°C and then pulsed in the same media supplemented with ³⁵S-labeled methionine and cysteine (Express Protein Labeling Mix, Perkin Elmer) at 200 μCi/ml for 15 min at 37°C. Cells were then washed with ice-cold RF10 and then incubated in RF10 at 37°C. At the selected timepoints, cells were washed in PBS and frozen. Cell pellets were lysed in 0.5% IGEPAL CA-630 (Sigma-Aldrich), 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂with Complete Protease Inhibitor Cocktail (Roche), and centrifuged 13,000 × g for 10 min to separate nuclei. Lysates were precleared twice with normal mouse serum (Sigma-Aldrich) and protein G–Sepharose and twice with protein G–Sepharose alone. MHC-I was immunoprecipitated using w6/32 antibody and protein G–Sepharose, and the immunoprecipitates were washed in NET buffer (0.5% IGEPAL CA-630, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA) three times. Precipitates were treated with Endoglycosidase Hf (New England Biolabs) according to the manufacturer's instructions. Proteins were denatured in reducing LDS-PAGE sample buffer and separated on NuPAGE 4–12% Bis-Tris precast gels (Life Technologies) before transferring onto PVDF membranes using the iBlot system (Life Technologies). Membranes were dried and exposed to a storage phosphor screen (GE Healthcare) and imaged on a Typhoon imager (GE Healthcare).

The purified CDHs were treated at 30 °C for 18 h with 3,200 U ml^–1 α-1,2/3-mannosidase and 50,000 U ml^–1 endoglycosidase H_f (New England Biolabs, Ipswich, MA, USA) in 50 mM sodium acetate buffer (pH 5.5) containing 5 mM ZnCl₂ to obtain 10 mg ml^–1 deglycosylated enzyme. To remove the glycosidases, column chromatography with Source 15Q was repeated as described above and the fractions containing pure CDH were pooled, diafiltered to 50 mM sodium acetate buffer (pH 5.5) and stored at 4 °C. Proteolytic cleavage in the linker of CDH was performed to obtain the individual DH and CYT domains. To this end, 40 μl of papain (10 mg ml^–1) was incubated at 25 °C for 1 h with 100 μl of activation buffer containing 2 mM EDTA and 2 mM dithiothreitol in 100 mM sodium phosphate (pH 7.0). CDH (final concentration 10 mg ml^–1) was digested in a reaction mix containing 140 μl per ml of the activated papain solution and 1 M sodium acetate (pH 5.0) at 25 °C for 4 h. The domains were separated from the residual intact CDH by column chromatography using a strong anion exchanger (Mono Q). The sample was diafiltered to 20 mM Tris-HCl (pH 8.0), loaded on the column and eluted by a linear NaCl gradient. Fractions containing the dehydrogenase and the cytochrome domain were pooled, concentrated and stored at 4 °C for further use.

Following treatment, cells were washed and harvested. Total cell lysates from U373 cell lines were subjected to SDS-PAGE followed by immunoblot analysis using the anti-gH mAb AP86 (gift from William J. Britt)29 (link), polyclonal anti-gL IgG, and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon, Billerica, MA). For proteasome inhibition experiments, cells were treated for 12 hours with the proteasome inhibitor ZL₃VS (2.5µM). For experiments involving Endoglycosidase H treatment, total cell lysates were incubated with 500 units of Endoglycosidase H_f (New England Biolabs) for 2 hours.