Nedd8

After drug incubation, neuroblastoma cells were lysed in 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mM PMSF, and 1 complete mini protease inhibitor cocktail (Roche). Immunoblot analysis was performed using the following antibodies: WEE1 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA, sc5285), Cdt-1 (1:200, Cell Signaling Technology, Danvers, MA, USA, 3386S), vinculin (1:200, Abcam, Cambridge, MA, USA, EPR8185), and NEDD8 (1:200, Cell Signaling Technology, Danvers, MA, USA, 2745S, and a generous gift from Takeda Pharmaceuticals, Tokyo, Japan). Immunoblot analysis was performed as previously described [22 (link)].

Immunoblot analysis was performed as described previously [47 (link)]. Briefly, cells were lysed in RIPA buffer and denatured at 95 °C for 5 min in Laemmli buffer. Samples were resolved on SDS–PAGE gels and transferred to PVDF membrane. Blots were probed with antibodies specific to ARIH1 (VPA00397; BioRad), hnRNP E1 (M01; Abnova), E-cadherin (3195; Cell Signaling), N-cadherin (BD Transductions), Vimentin (5741; Cell Signaling), Biotin (5597; Cell Signaling), Flag (14793; Cell Signaling), K48 Ubiquitin (8081; Cell Signaling), K63 Ubiquitin (5621; Cell Signaling), Nedd8 (2745; Cell Signaling), Hsp90 (sc13119: Santa Cruz Biotechnology), CD44 (GTX102111; GeneTex), V5 (R96025; ThermoFisher), UPF1 (VMA00627; BioRad), Filamin A (VMA00322; BioRad), Cortactin (VMA00430; BioRad), CAND1 (VMA00610; BioRad), and FASN (VMA00266; BioRad). Chemiluminescence was detected by CCD camera (BioRad ChemiDoc system).

Proteins were extracted using 1X RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA), and concentration determined using the BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA). Immunoblots (western blot) were performed as previously described [48 (link)]. Primary antibodies against ERK1/2, p-ERK1/2 (Thr202/Tyr204), MEK1/2, p-MEK1/2 (Ser217/221), p-PKCα/β II (Thr638/641), CAMKII, p-CAMKII (Thr286), BIM; p-BIM (Ser69), CHOP, Bcl-2, Bcl-xL, p-eIF2α (Ser51), STIM1, PARP, NEDD8 and secondary HRP-conjugated antibodies were obtained from Cell Signaling (Danvers, MA). Antibodies for PKCβ, luciferase, ERO1 and Orai1 were purchased from Santa Cruz Biotechnology (Dallas, TX). Proteins expression was determined by densitometry analysis of the immune-detected bands, normalized to β-actin, and presented relative to control (fold induction). Co-IP assays were carried out using the Pierce™ Classic Magnetic IP Kit (Thermo Fisher Scientific Inc., Waltham, MA) as described [49 (link)].

Human ESCC tissue arrays, which purchased from Shanghai Outdo Biotech Co. Ltd. (Shanghai, China), were immunohistochemically stained with antibody to NEDD8 (Cell Signaling, Boston, MA). The tissue array sections were dehydrated and subjected to peroxidase blocking, then incubated with anti-NEDD8 at room temperature. The tissue array sections were then stained with a GTVisionTM III Detection System/Mo&Rb (Gene tech Company Limited) and counterstained with hematoxylin. The histologic evaluation was based on calculation of the percentage of positive tumor cells and the staining intensity, as described previously^{22 (link)}. The detailed clinicopathologic characteristics of patients with ESCC are presented in Supplementary Table S1. This study was approved by the ethics committee of Huadong Hospital Affiliated to Fudan University (Approval No. 2016K007).

Pevonedistat was synthesized by Millennium Pharmaceuticals, Inc. The following reagents were purchased from their respective companies: recombinant rat TNF-α (Peprotech, Rocky Hill, NJ, USA); caspase inhibitors Z-VAD-FMK and Z-IETD-FMK (R&D Systems, Minneapolis, MN, USA); Necrostatin-1 (Sigma-Aldrich, St. Louis, MO, USA); and Epoxomicin (Sigma-Aldrich). Antisera were purchased from the following companies: β-Actin, cleaved caspase-3, cleaved caspase-8 (p18), cFLIP, cullin-3, IκBα, phospho-IκBα, NEDD8, PARP, pro-caspase-3, pro-caspase-6, pro-caspase-7, pro-caspase-8 (p10), pro-caspase-9 (Cell Signaling, Danvers, MA, USA); MLKL (Millipore, Billerica, MA, USA); CDT1 (Santa Cruz Biotechnology, Dallas, TX, USA); and BID (eBioscience, San Diego, CA, USA). Complete antisera details are provided in Supplementary Information.

All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at The Johns Hopkins University School of Medicine. Eight to ten weeks old C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Unless otherwise stated, all reagents were obtained from Sigma (St. Louis, MO). Antibodies to hemagglutinin (HA; C29F4), HDAC6, NEDD8, p-eNOS (T495) and FLAG were purchased from Cell Signaling (Danvers, MA). NOS3 (A-9) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX). Acetylated α-tubulin antibody was purchased from Abcam (ab24610; Cambridge, MA), total α-tubulin antibody was purchased from Invitrogen (62204; Waltham, MA), and Lipofectamine 2000 was purchased from Life Technologies (Waltham, MA). Tubacin was purchased from Enzo Life Sciences (Farmingdale, NY). Fresh batches of oxidized low-density lipoprotein (OxLDL) was purchased from Alfa Aesar (a Johnson Matthew company).

Antibodies against phospho-IκBα, phospho-IRF3, NEDD8, and UBA3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against IκBα and actin and NF-κB inhibitor JSH-23 (dissolved in dimethylsulphoxide, DMSO) were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibody against p65 was from Epitomics (Burlingame, CA). Antibody against IRF3 was from Abclonal (Cambridge, MA, USA). FBS was from HyClone Laboratories (Logan, UT, USA). M-CSF was from Cetus (Emeryville, CA, USA). TRIzol reagent, Moloney murine leukemia virus reverse transcriptase, and oligo(dT) primer were from Invitrogen (Carlsbad, CA, USA).