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Cell Signaling Technology Inc mouse monoclonal anti traf3
Mouse Monoclonal Anti Traf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse monoclonal anti traf3 - by Bioz Stars, 2026-10

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Article Title: Hematopoietic expression of cIAP2 drives inflammation and heart failure after myocardial infarction
Article Snippet: Bone marrow chimeric mice were established by lethally irradiatiating 6−8-week-old B6 CD45.1 mice with exposure to two cycles (spaced by 20-minute intervals) of 450-cGy gamma irradiation using a Gammacell 3000 Cesium 137 source irradiator (Best Theratronics). .. 300 μg of cell extracts was incubated in lysis buffer and 2 μg ml −1 mouse monoclonal anti-TRAF3 (Santa Cruz Biotechnology), rabbit ployclonal anti-TRAF6 (Cell Signaling Technology) or mouse monoclonal anti-RIPK1 (BD Biosciences) antibodies, respectively. .. Samples were rotated continuously at 4 °C overnight and subsequently incubated with 0.10 volumes of Protein G-conjugated Dynabeads (Thermo Fisher Scientific) at 4 °C for an additional 90 minutes.

Lysis:

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Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: a Workflow for fusion analysis in biliary tract cancer(BTC). b Schematic representation of PUM1-TRAF3 fusion, involving PUM1 (PUF domain, chromosome 1) and TRAF3 (MATH domain, not the RING and Zinc finger domains; chromosome 14). Schematic diagrams and circos plots were visualized using Arriba. In the index patient (patient 1 (B01 or P1)), the chimeric junction between PUM1 and TRAF3 for PUM1-TRAF3 fusion was confirmed via Sanger sequencing. c Fluorescent in situ hybridization (FISH) with two probes for PUM1 (red) and TRAF3 (green) in the P1 tissue slide. Arrow, fused signal. d In situ proximal ligation assay (PLA) was performed to visualize PUM1-TRAF3 protein expression in the P1 tissue slides using PUM1 rabbit polyclonal antibody and TRAF3 mouse monoclonal antibody; spot-like PLA signals were observed in the sample from the patient (B01 or P1) expressing the fusion protein (PUM1-TRAF3, red; DAPI, blue). e ddPCR product (P1, P5, P11, P12, and P36) was confirmed via Sanger sequencing. f FISH was performed with PUM1 (red) and TRAF3 (green) probes in the P5, P11, P12, and P36 tissue slides. Arrow, fused signal.

Article Snippet: Deparaffinized slides were incubated with a rabbit polyclonal antibody against PUM1 (Santa Cruz Biotechnology, Dallas, TX, USA) and a mouse monoclonal antibody against TRAF3 (Santa Cruz) in an antibody diluent (DakoCytomation California, Inc., Carpinteria, CA, USA).

Techniques: Sequencing, In Situ Hybridization, In Situ, Ligation, Expressing

$a Diagram of full-length PUM1, TRAF3 , and PUM1-TRAF3 (PT) fusion constructs. b PUM1-TRAF3 expression enhanced cell proliferation by 69.06 ± 1.90% ( p < 0.001) in SNU1196 cells and by 26.88 ± 0.77% (*** p < 0.001) in SNU308 cells. Data presented as mean ± S.D. c PUM1-TRAF3 expression elevates NIK and non-canonical NF-κB pathway associated proteins. The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. d PUM1-TRAF3 -induced NF-κB activation results in the translocation of NF-κB2(p52) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. e PUM1-TRAF3 interacts NIK: Protein-protein interactions among NIK, TRAF3, and truncated TRAF3 from PUM1-TRAF3 were assessed in 1196PT via immunoprecipitation. f PUM1-TRAF3 interacts TRAF2, TRAF3, and NIK: Protein-protein interactions among NIK, TRAF2, TRAF3, and PUM1 were assessed in 1196C and 1196PT via immunoprecipitation (IP, immunoprecipitation; IB, immunoblot). g Diagram of full-length TRAF3 , and partial TRAF3 constructs. h TRAF3 tail (TT)-transfected cells elevates NIK expression. Analysis NIK expression of partial TRAF3 -transfected cell proteins (CV, control vector; TH, TRAF3 head; TT, TRAF3 tail). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. i TRAF3 tail (TT)-transfected cells enhance the translocation of NF-κB2 ( p52 ) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (** p < 0.01; *** p < 0.001). Data presented as mean ± S.D.$

Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: a Diagram of full-length PUM1, TRAF3 , and PUM1-TRAF3 (PT) fusion constructs. b PUM1-TRAF3 expression enhanced cell proliferation by 69.06 ± 1.90% ( p < 0.001) in SNU1196 cells and by 26.88 ± 0.77% (*** p < 0.001) in SNU308 cells. Data presented as mean ± S.D. c PUM1-TRAF3 expression elevates NIK and non-canonical NF-κB pathway associated proteins. The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. d PUM1-TRAF3 -induced NF-κB activation results in the translocation of NF-κB2(p52) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. e PUM1-TRAF3 interacts NIK: Protein-protein interactions among NIK, TRAF3, and truncated TRAF3 from PUM1-TRAF3 were assessed in 1196PT via immunoprecipitation. f PUM1-TRAF3 interacts TRAF2, TRAF3, and NIK: Protein-protein interactions among NIK, TRAF2, TRAF3, and PUM1 were assessed in 1196C and 1196PT via immunoprecipitation (IP, immunoprecipitation; IB, immunoblot). g Diagram of full-length TRAF3 , and partial TRAF3 constructs. h TRAF3 tail (TT)-transfected cells elevates NIK expression. Analysis NIK expression of partial TRAF3 -transfected cell proteins (CV, control vector; TH, TRAF3 head; TT, TRAF3 tail). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. i TRAF3 tail (TT)-transfected cells enhance the translocation of NF-κB2 ( p52 ) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (** p < 0.01; *** p < 0.001). Data presented as mean ± S.D.

Techniques: Construct, Expressing, Activation Assay, Translocation Assay, Protein-Protein interactions, Immunoprecipitation, Western Blot, Transfection, Control, Plasmid Preparation

$a Both 1196C and 1196PT cells with silenced TRAF3 gene expression ( TRAF3 KO) showed increased proliferation (63.55 ± 3.65% in 1196C TRAF3 KO, 37.36 ± 1.22% to 68.72 ± 8.55% in 1196PT and 1196PT TRAF3 KO, respectively (*** p < 0.001). Data presented as mean ± S.D. b Proteins differentially expressed in PUM1-TRAF3 -transduced cells were analyzed; these included PUM1, TRAF3, PUM1-TRAF3, and NIK (*** p < 0.001). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. c PUM1-TRAF3-induced NF-κB activation with or without naïve TRAF3 results in the translocation of NF-κB2( p52 ) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. d Immunofluorescence staining for NF-κB2 and RelB in SNU1196 control vector-transduced and SNU1196 PUM1-TRAF3 -transduced cells. TRAF3 knockout enhances NF-κB2 and RelB translocation in control vector and PUM1-TRAF3 -transduced cells. (DAPI, blue; Green, PT or C; Red, NF-κB2 or RelB). e PUM1-TRAF3 -transduced and control vector (C)-transduced cells (5 × 10 6 cells/site) with or without TRAF3 expression were injected subcutaneously into four groups of mice, and tumor size was measured every 7 days (volume = length × width 2 × 1/2). At 27 days post injection, the average volumes of tumors were 582.6 ± 223.6 mm 3 , 802.8 ± 167.0 mm 3 , 898.9 ± 349.7 mm 3 ( p < 0.05), and 1019.9 ± 156.6 mm 3 ( p < 0.01) in 1196C, 1196PT, 1196C TRAF3 KO, and 1196PT TRAF3 KO mice, respectively (* P < 0.05; ** P < 0.01; ns no significance). Data presented as mean ± S.D. f Immunohistochemical staining of GFP (control vector or PUM1 - TRAF3 ), NIK, NF-κB2(p52) and RelB in mouse tumor tissues in C- and PT-expressing cell lines.$

Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: a Both 1196C and 1196PT cells with silenced TRAF3 gene expression ( TRAF3 KO) showed increased proliferation (63.55 ± 3.65% in 1196C TRAF3 KO, 37.36 ± 1.22% to 68.72 ± 8.55% in 1196PT and 1196PT TRAF3 KO, respectively (*** p < 0.001). Data presented as mean ± S.D. b Proteins differentially expressed in PUM1-TRAF3 -transduced cells were analyzed; these included PUM1, TRAF3, PUM1-TRAF3, and NIK (*** p < 0.001). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. c PUM1-TRAF3-induced NF-κB activation with or without naïve TRAF3 results in the translocation of NF-κB2( p52 ) and RelB (C, cytoplasmic fraction; N, nucleic fraction). The protein expression was assessed by area (*** p < 0.001). Data presented as mean ± S.D. d Immunofluorescence staining for NF-κB2 and RelB in SNU1196 control vector-transduced and SNU1196 PUM1-TRAF3 -transduced cells. TRAF3 knockout enhances NF-κB2 and RelB translocation in control vector and PUM1-TRAF3 -transduced cells. (DAPI, blue; Green, PT or C; Red, NF-κB2 or RelB). e PUM1-TRAF3 -transduced and control vector (C)-transduced cells (5 × 10 6 cells/site) with or without TRAF3 expression were injected subcutaneously into four groups of mice, and tumor size was measured every 7 days (volume = length × width 2 × 1/2). At 27 days post injection, the average volumes of tumors were 582.6 ± 223.6 mm 3 , 802.8 ± 167.0 mm 3 , 898.9 ± 349.7 mm 3 ( p < 0.05), and 1019.9 ± 156.6 mm 3 ( p < 0.01) in 1196C, 1196PT, 1196C TRAF3 KO, and 1196PT TRAF3 KO mice, respectively (* P < 0.05; ** P < 0.01; ns no significance). Data presented as mean ± S.D. f Immunohistochemical staining of GFP (control vector or PUM1 - TRAF3 ), NIK, NF-κB2(p52) and RelB in mouse tumor tissues in C- and PT-expressing cell lines.

Techniques: Gene Expression, Expressing, Activation Assay, Translocation Assay, Immunofluorescence, Staining, Control, Plasmid Preparation, Knock-Out, Injection, Immunohistochemical staining

$a Cells treated with DMSO or NIK inhibitor, 10 µM Amgen16, and nuclear and cytoplasmic fractions were isolated via cell compartment fractionation, and p52 and RelB were assessed (C, cytoplasmic fraction; N, nucleic fraction; *** P < 0.001; ns no significance). Data presented as mean ± S.D. b NIK inhibition diminishes PUM1-TRAF3-induced activation. NIK, NF-κB2, and RelB were visualized in C-transduced cells, TRAF3 knockout cells, PUM1-TRAF3-transduced cells, and TRAF3 knockout PUM1-TRAF3-transduced cells treated with DMSO or 1 µM Amgen16, via immunofluorescence staining. (DAPI, blue; green, PT or C; red, NIK, NF-κB2 or RelB).$

Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: a Cells treated with DMSO or NIK inhibitor, 10 µM Amgen16, and nuclear and cytoplasmic fractions were isolated via cell compartment fractionation, and p52 and RelB were assessed (C, cytoplasmic fraction; N, nucleic fraction; *** P < 0.001; ns no significance). Data presented as mean ± S.D. b NIK inhibition diminishes PUM1-TRAF3-induced activation. NIK, NF-κB2, and RelB were visualized in C-transduced cells, TRAF3 knockout cells, PUM1-TRAF3-transduced cells, and TRAF3 knockout PUM1-TRAF3-transduced cells treated with DMSO or 1 µM Amgen16, via immunofluorescence staining. (DAPI, blue; green, PT or C; red, NIK, NF-κB2 or RelB).

Techniques: Isolation, Fractionation, Inhibition, Activation Assay, Knock-Out, Immunofluorescence, Staining

PLA was performed to visualize PUM1-TRAF3 protein expression in the P1, 5, 11, 12, and 36 patient tissue slides using PUM1 rabbit polyclonal antibody and TRAF3 mouse monoclonal antibody (PUM1-TRAF3, red; DAPI, blue), and elevated expression of NIK along with p52 and RelB in the nucleus could be observed as PLA signals expressing areas upon immunohistochemical analysis. Magnified areas are marked in red, orange, yellow, and blue for PLA, NIK, NF-κB2, and RelB, respectively.

Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: PLA was performed to visualize PUM1-TRAF3 protein expression in the P1, 5, 11, 12, and 36 patient tissue slides using PUM1 rabbit polyclonal antibody and TRAF3 mouse monoclonal antibody (PUM1-TRAF3, red; DAPI, blue), and elevated expression of NIK along with p52 and RelB in the nucleus could be observed as PLA signals expressing areas upon immunohistochemical analysis. Magnified areas are marked in red, orange, yellow, and blue for PLA, NIK, NF-κB2, and RelB, respectively.

Techniques: Expressing, Immunohistochemical staining

a Representative images of NIK expression by immunohistochemistry in patients with BTC. NIK. NIK staining was scored as an intensity of NIK-positive (+) tumor cells: no expression: 0, low expression: 1+, moderate expression: 2+, and strong expression: 3+. 0 or 1+ was considered as NIK-weak whereas 2+ or 3 + IHC staining scores of NIK was considered NIK-strong group. b Relative frequency of NIK-weak ( n = 35) and NIK-strong ( n = 20) groups according to the PUM1-TRAF3 fusion status. c Kaplan–Meier analysis for survival according to PUM1-TRAF3 fusion in patients with BTC ( n = 55). Disease-free survival (DFS) and overall survival (OS) were not significantly different between fusion-negative ( n = 50) and fusion-positive group ( n = 5). d Kaplan–Meier analysis for survival according to NIK expression in patients with BTC ( n = 55). NIK-weak group ( n = 35) presented significantly prolonged survival in DFS and OS compared to NIK-strong group ( n = 20). e , f Forest plots for multivariate Cox proportional hazards analysis of the contribution of clinical factors to DFS and OS presented NIK-strong expression was significantly associated with poorer DFS and OS.

Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: a Representative images of NIK expression by immunohistochemistry in patients with BTC. NIK. NIK staining was scored as an intensity of NIK-positive (+) tumor cells: no expression: 0, low expression: 1+, moderate expression: 2+, and strong expression: 3+. 0 or 1+ was considered as NIK-weak whereas 2+ or 3 + IHC staining scores of NIK was considered NIK-strong group. b Relative frequency of NIK-weak ( n = 35) and NIK-strong ( n = 20) groups according to the PUM1-TRAF3 fusion status. c Kaplan–Meier analysis for survival according to PUM1-TRAF3 fusion in patients with BTC ( n = 55). Disease-free survival (DFS) and overall survival (OS) were not significantly different between fusion-negative ( n = 50) and fusion-positive group ( n = 5). d Kaplan–Meier analysis for survival according to NIK expression in patients with BTC ( n = 55). NIK-weak group ( n = 35) presented significantly prolonged survival in DFS and OS compared to NIK-strong group ( n = 20). e , f Forest plots for multivariate Cox proportional hazards analysis of the contribution of clinical factors to DFS and OS presented NIK-strong expression was significantly associated with poorer DFS and OS.

Techniques: Expressing, Immunohistochemistry, Staining

TRAF3 regulates NIK by binding and degrading it, whereas PUM1-TRAF3 binds to NIK but avoids degradation due to: 1) the lack of RING and zinc finger domains, and 2) reduction in TRAF2-TRAF3 complex formation by recruitment of TRAF2 by PUM1-TRAF3, thereby reducing its availability and rescuing NIK from degradation. NIK inhibitor can block the activation of the non-canonical NF-κB pathway by PUM1-TRAF3 expression.

Journal: NPJ Precision Oncology

Article Title: PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

doi: 10.1038/s41698-024-00654-2

Figure Lengend Snippet: TRAF3 regulates NIK by binding and degrading it, whereas PUM1-TRAF3 binds to NIK but avoids degradation due to: 1) the lack of RING and zinc finger domains, and 2) reduction in TRAF2-TRAF3 complex formation by recruitment of TRAF2 by PUM1-TRAF3, thereby reducing its availability and rescuing NIK from degradation. NIK inhibitor can block the activation of the non-canonical NF-κB pathway by PUM1-TRAF3 expression.

Techniques: Binding Assay, Blocking Assay, Activation Assay, Expressing

Figure 6. Paricalcitol restored TRAF3 levels in PBMCs from patients with CKD, in experimental kidney injury, and in cultured cells. (A) TRAF3 protein levels in PBMCs from patients with ESKD treated or not with paricalcitol were determined by Western blot. Number of patients: ﬁve to eight per group. *P,0.05 versus control; #P,0.05 versus ESKD cells. (B) TRAF3 mRNA levels in PBMCs from patients with ESKD treated or not with paricalcitol were determined by real-time PCR. Number of patients: ﬁve to eight per group. *P,0.05

Journal: Journal of the American Society of Nephrology

Article Title: TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease

doi: 10.1681/asn.2019111206

Figure Lengend Snippet: Figure 6. Paricalcitol restored TRAF3 levels in PBMCs from patients with CKD, in experimental kidney injury, and in cultured cells. (A) TRAF3 protein levels in PBMCs from patients with ESKD treated or not with paricalcitol were determined by Western blot. Number of patients: ﬁve to eight per group. *P,0.05 versus control; #P,0.05 versus ESKD cells. (B) TRAF3 mRNA levels in PBMCs from patients with ESKD treated or not with paricalcitol were determined by real-time PCR. Number of patients: ﬁve to eight per group. *P,0.05

Article Snippet: Precleared lysates were incubated with 2.5–5 mg mouse monoclonal anti-TRAF3 antibody (sc-6933; Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Cell Culture, Western Blot, Control, Real-time Polymerase Chain Reaction

Figure 7. TRAF3 overexpression mimics the actions of paricalcitol. TRAF3 overexpression was achieved in cultured cells using acti- vation TRAF3/CRISPR/Cas9 DNA plasmid. Cells were stimulated with recombinant human soluble TWEAK (100 ng/ml). In some ex- periments, cells were preincubated for 48 hours with 15 mmol/L paricalcitol before TWEAK stimulation. (A) TRAF3 mRNA levels are increased in cells transfected with CRISPR/Cas9 TRAF3 activation plasmid as evaluated by real-time PCR. (B) NF-kB2 pathway activation was assessed by Western blot of NF-kB2 p52 and the NF-kB2–regulated cytokine CCL-21A. (C and D) Gene expression of the proinﬂammatory factors (C)CCL-2, CCL-, and IL-6 or (D) CCL-21A and CCL-19 were evaluated by real-time PCR. Data expressed as mean6SEM of three to ﬁve independent experiments. Differences between intervention and control groups were assessed by Mann–Whitney test. *P,0.05 versus control; #P,0.05 versus TWEAK-treated cells.

Journal: Journal of the American Society of Nephrology

Article Title: TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease

doi: 10.1681/asn.2019111206

Figure Lengend Snippet: Figure 7. TRAF3 overexpression mimics the actions of paricalcitol. TRAF3 overexpression was achieved in cultured cells using acti- vation TRAF3/CRISPR/Cas9 DNA plasmid. Cells were stimulated with recombinant human soluble TWEAK (100 ng/ml). In some ex- periments, cells were preincubated for 48 hours with 15 mmol/L paricalcitol before TWEAK stimulation. (A) TRAF3 mRNA levels are increased in cells transfected with CRISPR/Cas9 TRAF3 activation plasmid as evaluated by real-time PCR. (B) NF-kB2 pathway activation was assessed by Western blot of NF-kB2 p52 and the NF-kB2–regulated cytokine CCL-21A. (C and D) Gene expression of the proinﬂammatory factors (C)CCL-2, CCL-, and IL-6 or (D) CCL-21A and CCL-19 were evaluated by real-time PCR. Data expressed as mean6SEM of three to ﬁve independent experiments. Differences between intervention and control groups were assessed by Mann–Whitney test. *P,0.05 versus control; #P,0.05 versus TWEAK-treated cells.

Article Snippet: Precleared lysates were incubated with 2.5–5 mg mouse monoclonal anti-TRAF3 antibody (sc-6933; Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Over Expression, Cell Culture, CRISPR, Plasmid Preparation, Recombinant, Transfection, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Gene Expression, Control, MANN-WHITNEY

Figure 8. Modulation of TRAF3 ubiquitination and cIAP1-TRAF3 complex formation was involved in paricalcitol restoration of TRAF3 levels. (A) In PBMCs from patients with ESKD on hemodialysis treated or not with paricalcitol (VDRA), TRAF3 was immunoprecipitated (I.P.) by an anti-TRAF3 antibody followed by SDS-PAGE and Immunoblotting (I.B.) using an anti-ubiquitin antibody. Representative experiment. TRAF3 antibody was used as loading control. cIAP1 levels were also evaluated. Number of patients ﬁve to eight per group. (B) TWEAK-induced TRAF3 ubiquitination was prevented by paricalcitol in HK2 cells stimulated with TWEAK and treated or not with paricalcitol. Figures show a representative I.P. experiment out of three performed.* IgG heavy chain.

Journal: Journal of the American Society of Nephrology

Article Title: TRAF3 Modulation: Novel Mechanism for the Anti-inflammatory Effects of the Vitamin D Receptor Agonist Paricalcitol in Renal Disease

doi: 10.1681/asn.2019111206

Figure Lengend Snippet: Figure 8. Modulation of TRAF3 ubiquitination and cIAP1-TRAF3 complex formation was involved in paricalcitol restoration of TRAF3 levels. (A) In PBMCs from patients with ESKD on hemodialysis treated or not with paricalcitol (VDRA), TRAF3 was immunoprecipitated (I.P.) by an anti-TRAF3 antibody followed by SDS-PAGE and Immunoblotting (I.B.) using an anti-ubiquitin antibody. Representative experiment. TRAF3 antibody was used as loading control. cIAP1 levels were also evaluated. Number of patients ﬁve to eight per group. (B) TWEAK-induced TRAF3 ubiquitination was prevented by paricalcitol in HK2 cells stimulated with TWEAK and treated or not with paricalcitol. Figures show a representative I.P. experiment out of three performed.* IgG heavy chain.

Article Snippet: Precleared lysates were incubated with 2.5–5 mg mouse monoclonal anti-TRAF3 antibody (sc-6933; Santa Cruz Biotechnology) overnight at 4°C.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, SDS Page, Western Blot, Control

Figure 2. ERK-controlled co-localization of endogenous HIF-1α with mitochondria

Journal: Journal of cell science

Article Title: Mortalin-mediated and ERK-controlled targeting of HIF-1α to mitochondria confers resistance to apoptosis under hypoxia.

doi: 10.1242/jcs.195339

Figure Lengend Snippet: Figure 2. ERK-controlled co-localization of endogenous HIF-1α with mitochondria

Article Snippet: The following antibodies were used: affinity purified rabbit polyclonal antibody against HIF-1α (Lyberopoulou et al., 2007); rabbit polyclonal antibodies against mortalin (sc-13967, 1:1000 dilution), TOM20 (sc-11415, 1:1000 dilution) and Gal4-DBD (sc-577, 1:500 dilution), mouse monoclonal antibody against C-terminus of PARP-1 (sc8007, 1:5000 dilution) or goat polyclonal antibody against VDAC1 (sc-8828, 1:500 dilution) all from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit polyclonal antibodies against phospho-ERK1/2 (9101, 1:1000 dilution), ERK1/2 (9102, 1:1000 dilution), HSP60 (4870, 1:1000 dilution), Caspase 3 (9662, 1:1000 dilution) and cytochrome c (4272, 1:500 dilution), rabbit monoclonal antibodies against HKII (6867, 1:1000 dilution) and cleaved Caspase-3 (9664, 1:500 dilution) and mouse monoclonal antibody against actin (3700, 1:5000 dilution) all from Cell Signaling (Danvers, MA, USA); mouse monoclonal antibodies against HIF-1α (610959, 1:1000 dilution),ARNT (611079, 1:500 dilution) and phosphoserine (612547, 1:1000 dilution) from BD Biosciences (San Jose, CA, USA), mouse monoclonal antibody against N-terminus of PARP-1 (ALX-804-211, 1:5000 dilution) from Enzo Life Sciences (Farmingdale, NY, USA), rabbit polyclonal antibody against HIF-2α (ΝΒ100-122, 1:1000 dilution) from Novus Europe (Cambridge, UK) or against Flag (F4042, 1:10000 dilution) from SigmaAldrich (St Louis, MO, USA).

Techniques:

Figure 3. ERK-controlled co-localization of GFP-HIF-1α with mitochondria under

Journal: Journal of cell science

Article Title: Mortalin-mediated and ERK-controlled targeting of HIF-1α to mitochondria confers resistance to apoptosis under hypoxia.

doi: 10.1242/jcs.195339

Figure Lengend Snippet: Figure 3. ERK-controlled co-localization of GFP-HIF-1α with mitochondria under

Techniques:

Figure 4. ERK-controlled and mortalin-mediated association of HIF-1α with the

Journal: Journal of cell science

Article Title: Mortalin-mediated and ERK-controlled targeting of HIF-1α to mitochondria confers resistance to apoptosis under hypoxia.

doi: 10.1242/jcs.195339

Figure Lengend Snippet: Figure 4. ERK-controlled and mortalin-mediated association of HIF-1α with the

Techniques:

Figure 5. Mitochondrial HIF-1α inhibits apoptosis when ERK is inactivated. (A) &

Journal: Journal of cell science

Article Title: Mortalin-mediated and ERK-controlled targeting of HIF-1α to mitochondria confers resistance to apoptosis under hypoxia.

doi: 10.1242/jcs.195339

Figure Lengend Snippet: Figure 5. Mitochondrial HIF-1α inhibits apoptosis when ERK is inactivated. (A) &

Techniques:

Figure 6. Mitochondrial HIF-1α inhibits drug-induced apoptosis under normoxia

Journal: Journal of cell science

Article Title: Mortalin-mediated and ERK-controlled targeting of HIF-1α to mitochondria confers resistance to apoptosis under hypoxia.

doi: 10.1242/jcs.195339

Figure Lengend Snippet: Figure 6. Mitochondrial HIF-1α inhibits drug-induced apoptosis under normoxia

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