Anti c flip

Western blot was performed as described [21 (link)]. Primary antibodies used were anti-actin (1:2000 MP Biomedicals C4), anti-caspase-8 (1:300 Cell Signaling #9746 and 1:300, Abcam ab32125), anti-caspase-3 (1:1000 Cell Signaling #9662), anti-caspase-7 (1:400 Cell Signaling #12827), anti-RIP1 (1:500 BD Biosciences 610458), anti-XIAP (1:400 BD Biosciences 610762), anti-cIAP1 (1:200 R&D Systems AF8181), anti-p100/p52 (1:500 Cell Signaling #4882) and anti-c-FLIP (1:400 Cell Signaling #5634). Secondary horseradish peroxidase-labeled antibodies were from GE Healthcare and Dako and were used at 1:5000 dilutions. For the chemiluminescent reaction, Supersignal Substrate (Thermo Scientific) was used. Chemiluminescence was detected with a LAS-1000 CCD camera and Image Reader LAS-1000 Pro v2.6 software (Fujifilm, Tokyo, Japan) or an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco/BRL Life Technologies (Grand Island, NY, USA). The anti-Bak, anti-Bcl-2, anti-PARP, anti-p53, anti-phospho-p53, anti-acetyl-p53, anti-Noxa, anti-PUMA, anti-TRAIL, anti-DR5, anti-C-Flip and anti-SIRT1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-p21 and anti-Myc antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The anti-acetyl-Myc and β-actin antibody were from EMD Millipore, Inc. (Burlington, MA, USA). The antisera to tNOX used in our western blot analyses were generated as described previously [18 (link)]. The anti-mouse and anti-rabbit IgG antibodies and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified.

Tetrandrine (TET) and dimethyl sulfoxide (DMSO) were bought from Sigma Chemical Co. (St. Louis, MO, USA), and TET was dissolved in dimethyl sulfoxide (DMSO) as 150 mg/mL stock. Hygromycin B was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Roswell Park Memorial Institute (RPMI) 1640 Medium and penicillin-streptomycin were purchased from Life Technologies (Carlsbad, CA, USA). Heat-inactivated fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT, USA), D-luciferin and pGL4.50 luciferase reporter (pGL4.50 [luc2/CMV]) vector from Promega (Madison, WI, USA), and JetPEI™ transfection reagent from Polyplus Transfection (Illkirch, Bas-Rhin, France). Primary monoclonal antibody anti-c-FLIP (1:300 dilution), anti-MCL-1 (1:300 dilution), anti-cleaved caspase-3 (1:300 dilution), anti-cleaved caspase-8 (1:300 dilution), and anti-cleaved caspase-9 (1:300 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-XIAP from Elabscience Biotechnology Inc. (1:300 dilution; Houston, TX, USA).

Western blotting was performed as previously described [57 (link)], using anti-AGK antibody (Epitomics, Burlingame, CA), anti-IKKβ, anti-p-IKKβ (Santa Cruz Biotech., Santa Cruz, CA), anti- IκBα, anti-p-IκBα, anti-c-FLIP, anti-XIAP, anti-Bcl-xl, anti-Bcl-2, anti-p65, anti-p84 antibodies (Cell Signaling, Danvers, MA). The membranes were stripped and re-probed with an anti-α-tubulin antibody (Sigma, Saint Louis, MI) as a loading control.

Human embryonic kidney T-REx-293/hemagglutinin (HA)-MCPIP1 and T-REx-293/HA-MCPIP1-D141N cell lines were kindly provided by Dr. Yi-Ling Lin (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan). The cells can be induced by tetracycline (Tet) to express HA-MCPIP1 or HA-MCPIP1-D141N and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Anti-cleaved caspases-3, -7, -8, and -9 as well as anti-phospho-extracellular signal-regulated kinase (ERK), anti-phospho-JNK, anti-phospho-p38, anti-phospho-IκBα, anti-phospho-IKKα/β, anti-IκBα, anti-cFLIP, anti-cIAP1, anti-HA, and anti-poly(ADP ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); the anti-GAPDH, anti-KPNA3, anti-KPNA4, and anti-MCPIP1 antibodies were obtained from GeneTex International (Hsinchu City, Taiwan); the anti-IKKα/β, anti-NF-κB p50, and anti-NF-κB p65 antibodies, and Protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and the anti-α-tubulin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA).

The anti-SIRT1 (# 2496), anti-Atg5 (# 2630), anti-ULK1 (# 6439), anti-LC3 (# 4108), anti-PARP (# 9542), anti-Bak (# 12105), anti-Bax (# 2772), anti-Puma (# 4976), anti-Noxa (# 14766), anti-Bcl2 (# 15071), anti-c-Flip (# 56343), anti-c-Myc (# 5605), HA-tag (# 3724), and anti-cleaved caspase-3 (# 9661) antibodies were purchased from Cell Signaling Technology, Inc (Beverly, MA, USA). The anti-acetyl-c-Myc (# ABE26) antibody was from Millipore Corp. (Temecula, CA, USA). The anti-Atg7 (# NB110-55474) antibody was obtained from Novus Biologicals (Centennial, CO, USA). The anti-NOX4 (# SC-30141) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The antisera to tNOX used for immunoblotting were generated as described previously (Chen et al., 2006 (link)). The commercially available anti-ENOX2 (# 10423–1-AP) antibody and anti-β-actin (# 60008–1-Ig) antibodies were from Proteintech (Rosemont, IL, USA) was used for immunoprecipitation. The anti-mouse (# 115-035-003) and anti-rabbit IgG (# 111-035-003) antibodies were purchased from the Jackson ImmunoResearch Laboratories Inc, (West Grove, PA, USA).

The following antibodies were from Cell Signaling Technology: anti-Casp8 (9746), anti-Cap3 (9664), anti-cIAP2 (3136), anti-pSAPK/JNK (JNK1/2, 4668), anti-pIκBα (9246), anti-Caveolin1 (3267), and anti-cFLIP (56343). The following antibodies were from Abcam: anti-pMLKL (187091), anti-RIPK3 (72106), anti-pRIPK3 (209384), and anti-FADD (108601). Anti-Ub (S.C8017), and protein A/G-conjugated beads were from Santa Cruz Biotechnology, anti-total MLKL (M6697) was from Sigma-Aldrich, anti-RIPK1 (610459) was from BD Medical Technology, and anti-cIAP1 was a gift from of John Silke (Walter and Eliza Hall Institute of Medical Research). z-VAD-fmk was from Enzo Life Sciences, RIPK1 inhibitor GSK’963, RIPK3 inhibitor GSK’840 and IAP antagonist SMAC007, as well as the pRIP1 S166-specific antibody, were provided by GlaxoSmithKline^{34 (link)}. IAP antagonist BV6 was provided by Domogoj Vucic (Genentech), recombinant human TNF was from R&D or from PeproTech, Flag-Tagged TNF was from Enzo, necrosulfonamide was from CalBiochem, TLCK (Tosyl-L-lysyl-chloromethane hydrochloride) was from Abcam, cycloheximide (CH) was from Sigma-Aldrich, and Carfilzomib was from BioVision.