Journal: Journal of Biological Chemistry

Article Title: X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factor β1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)

doi: 10.1074/jbc.m505671200

Figure Lengend Snippet: FIGURE 5. XIAP mediates the negative cross-talk between the NF-B and JNK pathways. A, NMH cells were transfected with 50 nM siRNA specific for murine XIAP (siXIAP) or scrambled siRNA control (siC). After 24 h, siRNA-transfected cells were treated for the indicated times with 5 ng/ml TGF-1. WCEs were subjected to immunoblotting using antibodies against XIAP, phospho-Jun, c-Jun, or actin. B, NMH cells were transfected with siXIAP or siC as described above and WCEs isolated in kinase buffer. WCEs (300 g) were immunopre- cipitated (IP) with an antibody against JNK1 and an aliquot was subjected to a kinase assay using GST-Jun as substrate. An equal aliquot of each immunoprecipitant was subjected to immunoblotting, as indicated. C and D, NMH cells were plated in triplicate in 96-well plates and transfected by lipofection with 50 ng of 4XTRE-Lux (C) or 3TP-Lux (D) constructs in the presence or absence of wild-type XIAP (wtXIAP), XIAP-BIR, XIAP-RING, or TAK1-K63W and an internal control Renilla luciferase expression plasmid. Following 6 h of treatment with 5 ng/ml TGF-1, luciferase activity was measured and expressed as fold induction relative to that of BSA-treated cells that was set at 1. Means and standard deviations are representative of two independent experiments carried out in triplicate. Inset, NMH cells were transfected for 24 h with Myc-tagged wtXIAP, XIAP-BIR, XIAP-RING. WCEs were subjected to immunoblot analysis using an anti-Myc antibody. IB, immunoblot.

Article Snippet: The antibody preparations for IKK-2 (sc-7607), c-Jun (sc-45), phosphoJun (sc-822), TAK1 (sc-7967), HA (sc-805), and Myc (sc-40) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Control, Western Blot, Isolation, Kinase Assay, Construct, Luciferase, Expressing, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factor β1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)

doi: 10.1074/jbc.m505671200

Figure Lengend Snippet: FIGURE 6. TGF-1 induces both the IKK and JNK signaling pathways. A, NMH cells were transfected with vectors directing expression of N-terminally FLAG-tagged TAK1 (TAK1) or the dominant negative FLAG-TAK1-K63W (TAK1-K63W). After 24 h, transfected cells were treated for the indicated times with 5 ng/ml TGF-1. WCEs (300 g) were immunoprecipi- tated (IP) with an antibody against TAK1, and an aliquot was subjected to a kinase assay using GST-MKK4/7 as substrate. An equal aliquot of each immunoprecipitant was subjected toimmunoblottingusingantibodiesspecificforTAK1,FLAG,phospho-Smad2(pSmad2),Smad2,andactinasindicated.B,NMHcellsweretransfectedandtreatedasdescribedabove. WCEs (40 g) were subjected to immunoblotting using antibodies raised against phospho-IKK1/2 (pIKK1/2), IKK1/2, Flag, or actin, as indicated. C, NMH cells transfected with 50 ng of b-Lux or 4XTRE-Lux construct in the presence or absence of the constitutive active form of the TRI T204D and an internal control Renilla luciferase expression plasmid. Cells not expressing the TRI T204D construct were stimulated for 6 h with 5 ng/ml TGF-1. Luciferase activity was measured and expressed as fold induction relative to that of BSA-carrier solution-treated cells that was set at 1. Means and standard deviations are representative of two independent experiments carried out in triplicate. IB, immunoblot.

Article Snippet: The antibody preparations for IKK-2 (sc-7607), c-Jun (sc-45), phosphoJun (sc-822), TAK1 (sc-7967), HA (sc-805), and Myc (sc-40) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Protein-Protein interactions, Transfection, Expressing, Dominant Negative Mutation, Kinase Assay, Western Blot, Construct, Control, Luciferase, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factor β1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)

doi: 10.1074/jbc.m505671200

Figure Lengend Snippet: FIGURE 7. XIAP promotes TAK1 degradation in response to TGF-1 treatment. A, NMH cells were treated for the indicated times with 5 ng/ml TGF-1. WCEs (300 g) were immunoprecipitated (IP) with a monoclonal anti-XIAP antibody or IgG control. Immuno- blotting of equal immunoprecipitated aliquots was performed using an anti-TAK1 or an anti-XIAP polyclonal antibody. B and C, WCEs of TGF-1-treated NMH cells (B) or primary cultures of rat hepatocytes (C) were subjected to immunoblotting using an antibody specific for either TAK1 or actin. D, NMH cells were treated for 12 h with 5 ng/ml TGF-1 alone or in combination with 20 M proteasome inhibitor MG132. WCEs were subjected to immunoblotting as described above. E, NMH cells were transfected with 50 nM siRNA specific for murine XIAP (siXIAP) or 50 nM nonspecific siRNA control (siC). Si-transfected cells were then treated with TGF-1 for the indicated times. WCEs were subjected to immunoblotting using antibodies against TAK1, XIAP, or actin. IB, immunoblot.

Article Snippet: The antibody preparations for IKK-2 (sc-7607), c-Jun (sc-45), phosphoJun (sc-822), TAK1 (sc-7967), HA (sc-805), and Myc (sc-40) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunoprecipitation, Control, Western Blot, Transfection

Journal: Journal of Biological Chemistry

Article Title: X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factor β1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)

doi: 10.1074/jbc.m505671200

Figure Lengend Snippet: FIGURE8.XIAPplaysaroleinTAK1ubiquitinationinresponsetoTGF-1treatment. A, NMH cells were transfected with FLAG-tagged TAK1 (pCMV2-FLAG-TAK1), His-tagged ubiquitin (pCW7-H6M-Ub), and Myc-tagged XIAP (pCS3-Myc-XIAP) in the presence of 20 M MG132. WCEs (300 g) were immunoprecipitated with anti-FLAG antibody or IgG control and subjected to immunoblotting (IB) using an anti-ubiquitin antibody. Equal immunoprecipitated aliquots were also subjected to immunoblotting using an anti- FLAG antibody (bottom panel, short exposure). B, NMH cells were transfected with FLAG- tagged TAK1 (Flag-TAK1), His-tagged ubiquitin (pCW7H6M-Ub), Myc-tagged XIAP (Myc- XIAP) Myc-XIAP-BIR, and Myc-XIAP-RING in the presence of 20 M MG132. FLAG immunoprecipitants were analyzed as described above. Bands relative to ubiquitinated TAK1 (ub-TAK1) were quantified by densitometric analysis and expressed in arbitrary units (n 2).

Article Snippet: The antibody preparations for IKK-2 (sc-7607), c-Jun (sc-45), phosphoJun (sc-822), TAK1 (sc-7967), HA (sc-805), and Myc (sc-40) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: X-linked Inhibitor of Apoptosis (XIAP) Inhibits c-Jun N-terminal Kinase 1 (JNK1) Activation by Transforming Growth Factor β1 (TGF-β1) through Ubiquitin-mediated Proteosomal Degradation of the TGF-β1-activated Kinase 1 (TAK1)

doi: 10.1074/jbc.m505671200

Figure Lengend Snippet: FIGURE 10. Model for the negative cross-talk between the NF-B and JNK signaling pathways during TGF-1-induced apoptosis. In response to TRI signaling, TAK1 acti- vates both IKKs and JNKs. Activation of JNKs promotes AP-1/Smads-mediated apoptosis. In contrast, activation of the IKK/NF-B axis results in protection from TGF-1 cell killing through regulation of XIAP gene expression. The proposed mechanism of this negative cross-talk between the NF-B and JNK pathways relies on the ability of XIAP to promote ubiquitin-mediated degradation of TAK1 in the 26 S proteasome thereby leading to the shutdown of JNK activity and rescue from cell death.

Article Snippet: The antibody preparations for IKK-2 (sc-7607), c-Jun (sc-45), phosphoJun (sc-822), TAK1 (sc-7967), HA (sc-805), and Myc (sc-40) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Protein-Protein interactions, Activation Assay, Gene Expression, Ubiquitin Proteomics, Activity Assay

Journal: Journal of Virology

Article Title: Disruption of MDA5-Mediated Innate Immune Responses by the 3C Proteins of Coxsackievirus A16, Coxsackievirus A6, and Enterovirus D68

doi: 10.1128/jvi.00546-17

Figure Lengend Snippet: Figure 12. 3Cpro from CA16, CV-A6, and EV-D68 targets TAK1 to impair the NF- κB response. 971

Article Snippet: The following antibodies were used for western blotting analysis in 147 this study: anti-Flag monoclonal M2 antibody (F1804-1MG; Sigma), anti-Myc (9E10) 148 monoclonal antibody (MMS-150P; Covance), anti-HA monoclonal antibody (MMS-101R; 149 Covance), anti-Tubulin monoclonal antibody (MMS-410P; Covance), anti-V5 antibody (V8012, 150 Sigma), VP1 antiserum against CA16 obtained from rabbits immunized with CA16 CC024, 151 RIG-I (D14g6) rabbit antibody (#3743; Cell Signal), Cardif (human) antibody (AT107) (ALX-152 210-929-C100; Enzo Life Sciences), MDA5 (human) polyclonal antibody (AT113) (ALX-210-153 935-C100; Enzo Life Sciences), TAK1 antibody (2E10; NOVUS) and anti-p-IRF3 monoclonal 154 antibody (AB76493; Abcam).

Techniques:

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) Volcano plot of m 6 A-IP-seq targets in shALKBH1 #1 As-Tr cells with NR2C2 as a top target labeled (black arrow). (B) HOMER motif analysis demonstrating the canonical m 6 A GGACU motif is enriched across experimental samples. (C) IGV visualization of increased m 6 A enrichment on representative site on the NR2C2 transcript. (D) qPCR analysis of the m 6 A levels on the NR2C2 transcript following m 6 A-IP in shNC and shALKBH1 #1 As-Tr cells. (E) qPCR analysis of the m 6 A levels on the NR2C2 transcript following m 6 A-IP of HaCaT control cells without or without 200 nM arsenic for 72 h and As-Tr cells. (F) CLIP-qPCR analysis of FLAG (ALKBH1) binding to sites on the NR2C2 transcript ( NM_001291694.2 , site 1: regions 1508243–1504892, and site 2: 150486–15048792, respectively) with an anti-FLAG or immunoglobulin G (IgG) antibody in shALKBH1 #1 As-Tr cells transfected with a construct expressing ALKBH1 (FLAG). (G) Immunoblot analysis of NR2C2 in shNC and shALKBH1 #1 As-Tr cells. (H) RT-qPCR of NR2C2 mRNA levels in shNC and shALKBH1 #1 As-Tr cells. (I) Immunoblot analysis of NR2C2 expression in HaCaT and A431 SCC cells. (J) Immunoblot analysis of NR2C2 expression in control (no As) and As-Tr cells. (K) Immunoblot analysis of NR2C2 expression in HaCaT cells treated with 200 nM arsenic for 72 h. (L) Immunoblot analysis of HA and NR2C2 expression in shALKBH1 #1 As-Tr cells expressing empty vector (EV), WT ALKBH1, catalytically inactive mutant ALKBH1 (D233A), and C118A and C207A mutant ALKBH1. (M) Immunoblot analysis of METTL3, METTL14, and NR2C2 expression in shALKBH1 #1 As-Tr cells transfected with control or METTL3/METTL14 siRNA. (N) Immunoblot analysis of YTHDF1 and NR2C2 expression in shALKBH1 #1 As-Tr cells transfected with control or YTHDF1 siRNA. (O) RNA immunoprecipitation (RIP) and qPCR analysis showing the binding of YTHDF1 to the NR2C2 transcript in As-Tr cells. (P) CCK8 assay of shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. (Q) Soft agar cloning assay of shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. (R) CCK8 assay of As-Tr cells with or without NR2C2 overexpression. (S) Soft agar cloning assay of As-Tr cells with or without NR2C2 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t test.

Article Snippet: NR2C2 ( NM_003298 ) Human Tagged ORF Clone , Origene , Cat# RC219184.

Techniques: Labeling, Control, Binding Assay, Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Mutagenesis, RNA Immunoprecipitation, CCK-8 Assay, Knock-Out, Cloning, Over Expression

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) SUnSET analysis of global translation levels across no As, Chr As, and As-Tr cells. (B) SUnSET analysis of global translation levels across HaCaT cells with ALKBH1 knockdown with or without arsenic treatment (200 nM, 72 h). (C) SUnSET analysis of global translation levels in As-Tr cells with ALKBH1 knockdown. (D) SUnSET analysis of global translation levels in shALKBH1 #1 As-Tr cells transfected with control or NR2C2 siRNA. (E) Immunoblot analysis of p-4EBP1 and 4EBP1 (total) in As-Tr cells with ALKBH1 knockdown. (F) Log2 fold change of SESN1 , SESN2 , and SESN3 mRNA expression in shALKBH1 #1 As-Tr cells as compared to shNC. Data taken from RNA-sequencing used as input for m 6 A-IP-sequencing. (G–I) IGV visualization of NR2C2 binding on SESN1 , SESN2 , and SESN3 transcripts taken from NR2C2 ChIP-seq data (ENCSR454GVT, ENCSR750LYM, ENCSR559ZKI) from ENCODE. (J) Immunoblot analysis of SESN1 and ALKBH1 expression in shNC and shALKBH1 #1 As-Tr cells. (K) Immunoblot analysis of p-4EBP1, 4EBP1 (total), SESN1, and NR2C2 expression in shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. **** p < 0.0001; Student’s t test.

Article Snippet: NR2C2 ( NM_003298 ) Human Tagged ORF Clone , Origene , Cat# RC219184.

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, RNA Sequencing, Sequencing, Binding Assay, ChIP-sequencing, Knock-Out