Journal: Experimental cell research

Article Title: Inhibition of inducible NF-kappaB activity reduces chemoresistance to 5-fluorouracil in human stomach cancer cell line.

doi: 10.1016/s0014-4827(03)00223-4

Figure Lengend Snippet: Fig. 3. NF-B DNA-binding activity and transcriptional activity in 5-FU-treated cells. (A) EMSA. Cells were treated with 10 M 5-FU for 5, 10, or 20 h as indicated. Both cell lines had constitutive NF-B activation, although inducible activation of NF-B was detected only in NUGC3/5FU/L cells after 10 h of 5-FU treatment. Control represents untreated cells. The results are representative of three separate experiments. (B) Supershift assay. Preincubation of nuclear extracts from NUGC3/5FU/L cells treated or untreated with anti-p65, p50, or cRel antibody showed that p65 and p50 were the predominant subunits of inducible NF-B after 10 h treatment of 10 M 5-FU. (C) Transcriptional activity of NF-B measured by luciferase reporter gene assay. Cells were transfected with the NF-B promoter/luciferase construct and then treated with 10 M 5-FU for 5, 10, or 20 h, followed by examination of luciferase activity. Control represents untreated cells. The Promega Dual-Luciferase Reporter Assay system was used according to the protocol provided by the manufacturer. Sample luciferase activity was normalized to control luciferase activity. Increased luciferase activity was detected in NUGC3/5FU/L but not in NUGC3 cells after 10 h of treatment with 5-FU (mean SD of three independent experiments).

Article Snippet: Identification of the distribution of primary antibody (antiNF- B p65 antibody; Santa Cruz Biotechnology, Santa Cruz, CA) was achieved by subsequent application of a biotinylated anti-primary and streptavidin-peroxidase.

Techniques: Binding Assay, Activity Assay, Activation Assay, Control, Luciferase, Reporter Gene Assay, Transfection, Construct, Reporter Assay

Journal: Journal of dairy science

Article Title: Effect of endoplasmic reticulum stress on metabolic and stress signaling and kidney-specific functions in Madin-Darby bovine kidney cells.

doi: 10.3168/jds.2016-12406

Figure Lengend Snippet: Figure 3. Effect of 24-h treatment with different concentrations of (A) tunicamycin (TM; 0, 0.1, and 1 µg/mL) or (B) thapsigargin (TG; 0, 0.1, and 1 µmol/L) on relative mRNA levels of nuclear factor-κB (NF-κB) and NOD-like receptor P3 (NLRP3) inflammasome target genes caveolin (CAV1), interleukin 1 β (IL1B), tumor necrosis factor (TNF), and PYD and CARD domain containing (PYCARD) in Madin-Darby bovine kidney (MDBK) cells. Cells treated with dimethyl sulfoxide (0.5% for TM and 0.1% for TG) as vehicle served as the control (0 µg/mL of TM, 0 µmol/L of TG). Representative gel blots depicting protein level of NF-κB subunit p50 in nuclear extracts of MDBK cells after 24-h treatment with (C) 1 µg/mL of TM or (D) 1 µmol/L of TG. Abundance of β-actin protein served as a loading control. Bars represent relative protein level expressed as fold of the control (0 µg/mL of TM, 0 µmol/L of TG) and are means ± SD from 3 independent experiments. *P < 0.05.

Article Snippet: After that, the membranes were washed and blocked for 1 h at room temperature with 5% nonfat dry milk (wt/vol) in Tris-buffered saline with 0.1% Tween 20 following overnight incubations with primary antibodies against NF-κB/p50 (rabbit polyclonal antiNF-κB/p50 antibody H-119; Santa Cruz Biotechnology, Heidelberg, Germany), total PERK (goat polyclonal anti-PERK antibody T-19; Santa Cruz Biotechnology), phospho-PERK [pPERK; rabbit monoclonal antipPERK(Th980) antibody; Cell Signaling Technology, Danvers, MA], and β-actin (mouse monoclonal anti-βactin antibody ab6276, Cambridge, UK) at 4°C as a reference protein for adequate normalization.

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: In TNF-stimulated Cells, RIPK1 Promotes Cell Survival by Stabilizing TRAF2 and cIAP1, which Limits Induction of Non-canonical NF-κB and Activation of Caspase-8

doi: 10.1074/jbc.m110.216226

Figure Lengend Snippet: FIGURE 5. TNF induces non-canonical NF-B in RIPK1/ MEFs. A, wild- type and RIPK1/ MEFs were treated with 100 ng/ml Fc-TNF for the times shown.Inparallel,wild-typeMEFswerealsotreatedwith500nMcompoundA for the same times. Cells were lysed in DISC buffer and analyzed for the levels of NIK, cIAP1, and TRAF2, and processing of p100 to p52. B, wild-type and RIPK1/ MEFs were treated with 100 ng/ml of Fc-TNF for the indicated times followed by fractionation into cytosolic and nuclear fractions. Fractions were then analyzed by Western blot for the levels of p100/p52 and Lamin A/C and Hsp70 as a loading controls.

Article Snippet: Reagents and Antibodies—The primary antibodies used were anti- -actin (A-1978, Sigma), anti-RIPK1 (610458, BD Transduction Laboratories), anti-cFLIP (XA-1008, ProScience), antiTRAF2 (sc-876, Santa Cruz Biotechnology), anti-cIAP1 (in house), anti-PARP (9542, Cell Signaling Technology), anticaspase-8 (Gift from Lorraine O’Reilly, Walter and Eliza Hall Institute), anti-NIK (4994, Cell Signaling Technology), antiNF- B2 P100 (4882, Cell Signaling Technology), anti-Lamin A/C (20681, Santa Cruz Biotechnology).

Techniques: Fractionation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: In TNF-stimulated Cells, RIPK1 Promotes Cell Survival by Stabilizing TRAF2 and cIAP1, which Limits Induction of Non-canonical NF-κB and Activation of Caspase-8

doi: 10.1074/jbc.m110.216226

Figure Lengend Snippet: FIGURE 9. Model of pro-death and pro-survival functions of RIPK1. A, wild-type cells. Step 1, TNF binding triggers assembly of complex I by recruitment of TRAFs and cIAPs, which results in p65/RelA NF-B translocation to the nucleus. Step 2, cFLIPL is up-regulated by NF-B. Step 3, in the presence of RIPK1, TRAF2, and cIAP1 are stabilized (potentially by direct interaction with RIPK1, but it does not require RIPK1 kinase activity) and TRAF2 mediates stabilization of cFLIPL by an unknown mechanism. Step 4, cFLIPL binds to caspase-8, blocking its activation and preventing apoptosis. B, pro-survival effect of RIPK1. Step 1, TNF binding triggers assembly of complex I by recruitment of TRAFs and cIAPs which results in NF-B translocation to the nucleus. Step 2, cFLIPL is up-regulated by NF-B. Step 3, in the absence of RIPK1, cIAP1 and TRAF2 are degraded by a proteasomal/lysosomal mechanism. This also leads to NIK stabilization and p100 processing (not shown), and translocation of p52/RelB dimers to the nucleus. Step 4, loss of TRAF2 leads to destabilization of cFLIPL, the majority of which is degraded by a proteasomal mechanism. A proportion of cFLIPL interacts with caspase-8 and is cleaved to p43cFLIP, thereby blocking full caspase-8 activation. Step 5, partial inhibition of transcription or translation reduces levels of cFLIPL and p43cFLIP allowing caspase-8 activation, resulting in apoptosis.

Article Snippet: Reagents and Antibodies—The primary antibodies used were anti- -actin (A-1978, Sigma), anti-RIPK1 (610458, BD Transduction Laboratories), anti-cFLIP (XA-1008, ProScience), antiTRAF2 (sc-876, Santa Cruz Biotechnology), anti-cIAP1 (in house), anti-PARP (9542, Cell Signaling Technology), anticaspase-8 (Gift from Lorraine O’Reilly, Walter and Eliza Hall Institute), anti-NIK (4994, Cell Signaling Technology), antiNF- B2 P100 (4882, Cell Signaling Technology), anti-Lamin A/C (20681, Santa Cruz Biotechnology).

Techniques: Binding Assay, Translocation Assay, Activity Assay, Blocking Assay, Activation Assay, Inhibition