N ethylmaleimide nem

APAP was obtained from Adamas-beta (Shanghai, China). Reagents used for TrxR activity determination: 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) and nicotinamide adenine dinucleotide phosphate (NADPH) were from Biosharp (Anhui, China); Aurothioglucose (ATG) was purchased from Wako (Osaka, Japan). Glutathione reductase (GR) and glutathione (GSH) were purchased from Solarbio (Beijing, China). N-ethylmaleimide (NEM) was purchased from Sigma-Aldrich (Saint Louise, MO, USA). Anti-GSH antibody (from mice) was purchased from Abcam (Cambridge, UK), anti-thioredoxin1 (Trx1), and anti-Trx2 antibodies were from IMCO (Stockholm, Sweden); all other antibodies used in the study were obtained from Proteintech (Hubei, China). Sep-Pak C18 cartridge was purchased from Waters Corporation (Milford, MA, USA).

pMyc-NS4B and pHA-Ub were cotransfected into HEK293T cells with or without pFlag-pRNF114. The cells were then treated with 10 μM MG132 (Sigma-Aldrich) for 24 h and were lysed by NP-40 lysis buffer containing a deubiquitinase inhibitor (10 μM N-ethylmaleimide [NEM]; Sigma-Aldrich). Then the anti-Myc MAb-conjugated agarose beads were added into the samples separately. Following incubation overnight at 4°C, the samples were examined by Western blotting.
For the in vitro ubiquitination assay, bacterially expressed GST fusion proteins of pRNF114 and pRNF114(C64/67A) were captured on glutathione-Sepharose beads and incubated for 90 min at 30°C with purified Ub-activating enzyme UBE1, His-tagged Ub-conjugating enzyme UbcH5A, Flag-Ub, and Ub conjugation reaction buffer (R&D Systems). The ubiquitin ligase activity of pRNF114 was analyzed by Western blotting with an anti-Flag MAb. For in vitro ubiquitination of NS4B by pRNF114, purified NS4B and pRNF114 were incubated with UBE1, UbcH5A, His-Ub, His-K27, or His-K27R in the presence of ATP. The in vitro ubiquitination of NS4B was analyzed by Western blotting.

Primary antibodies targeting the following proteins were used in this study: p-eIF2α, IRE-1α, CHOP (Cell Signaling Technologies, Danvers, MA, USA), GRP78, ATF6α, PERK, p-PERK, sXBP-1, β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), p-IRE-1α (Abcam, Cambridge, MA, USA) and monoclonal PDI (clone 1D3, Enzo Life Sciences, Farmingdale, NY, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. IC87114 was obtained from Calbiochem (San Diego, CA, USA). Ovalbumin, lipopolysaccharide, N-ethylmaleimide (NEM), 4-hydroxy-2-nonenal (4-HNE) and dihydroethidine (DHE) were purchased from Sigma-Aldrich (St Louis, MO, USA).

The following mouse monoclonal antibodies were used: anti-V5 (Invitrogen), anti-V5-conjugated agarose beads (Sigma–Aldrich), anti-His (GE Healthcare) and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Ambion). The rabbit polyclonal antisera raised against P5, PDI and ERp57 have been described previously [26 (link)]. N-ethylmaleimide (NEM) and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) were purchased from Sigma and Life technologies respectively.

Cells were stimulated with 50 ng/ml Biotin-TNF (R&D systems) for each indicated time point. For “0” time points, Biotin-TNF was added to cleared cell lysates. Two 15 cm plates of cells at 80% confluency were used for each condition. Media was replaced with 10 mL fresh media prior to lysis to remove unbound TNF. Cells were lysed in buffer containing 30 mM TrisHCl, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1% Triton X-100 supplemented with 1 mM DTT, 5 mM N-Ethylmaleimide (NEM; Sigma Aldrich), cOmplete protease inhibitors and phosSTOP. Lysates were incubated on ice for 15 min and cleared by centrifugation. Lysate samples were collected and for ”0” time point samples, Biotin-TNF was added. Lysates were subsequently incubated with Streptavidin magnetic beads (Thermo Fisher Scientific; 88816) for at least 2 h at 4°C and washed five times in lysis buffer. Samples were eluted with 1x LSB for 5 min at 95°C. For phosphatase treatment, beads were resuspended and washed prior to LSB elution in 1x protein metallophosphatase (PMP) buffer (New England Biolabs) supplemented with MnCl₂ and incubated for 30 min at 30°C with lambda protein phosphatase (λPP, New England Biolabs). Samples were analyzed by SDS-PAGE and immunoblotting.

Transfected cells were lysed in 500 μl of lysis buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton, 1 × Protease Inhibitor cocktail (Roche Applied Science), 50 mM N-ethylmaleimide (NEM, from Sigma)] and collected for centrifugation at 14,000 × g for 10 min. The supernatant was mixed with 25 μL of GFP-Nanotrap beads (Chromotek GmbH) that had been pre-washed with dilution buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 × Protease Inhibitor cocktail, 50 mM NEM). The mixture was incubated at RT for 150 min with gentle rolling and centrifuged for 2,700 × g for 2 min. The supernatant was removed and the beads washed once with dilution buffer, three times with washing buffer (8 M Urea, 1% SDS in PBS) and once with 1% SDS in 1× PBS. The bound proteins were eluted with sample loading buffer (250 mM Tris–HCl pH 7.5, 40% glycerol, 4% SDS, 0.2% BPB) by heating at 95 °C for 10 min. Eluted samples were run into 4–15% Tris–Glycine gels.

4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlorine (AEBSF, A8456), aprotinin (A1153), bestatin (Sigma B8385), pepstatin A (P5318), N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64, E3132), leupeptin (L2884), N-ethylmaleimide (NEM, E3876), calpeptin (C8999), calpain inhibitor-I (A6185), -II (A6060), -III (M6690), pan-caspase inhibitor (V116), BAPTA-AM (A1076), ionomycin (I0634) were supplied by Sigma. Calpistatin peptide (208902), negative control peptide (208904), elastase inhibitor IV (324759) and cathepsin G inhibitor I (219372) were supplied by Calbiochem. Tryptase inhibitor nafamostat mesylate (NM, 3081) was supplied by Tocris. AEBSF, aprotinin, bestatin, pepstatin A, E64, leupeptin and NEM were used in vitro at concentrations according to manufacturer’s instructions (Sigma Inhib1). calpeptin, calpain, NE, CG, caspase and tryptase inhibitors, BAPTA-AM were used at 100 μM during in vitro studies. Calpistatin and control peptide were used at 10 μM during in vitro studies. Doses of inhibitors used in vivo and details of all other reagents used are described in other method sections.