Journal: Journal of Applied Physiology

Article Title: Impaired exercise capacity and skeletal muscle function in a mouse model of pulmonary inflammation

doi: 10.1152/japplphysiol.00607.2012

Figure Lengend Snippet: NF-κB p65 subunit DNA binding activity is increased in female SP-C/TNFα+ skeletal muscle. DNA binding activity of the p65 NF-κB subunit was detected in gastrocnemius homogenates. Values represent means ± SE; n = 6. *P < 0.001 compared with all other groups.

Article Snippet: NFκB p65 DNA binding activity was measured using an ELISA-based kit (TransAM NF-κB p65, no. 40096 Active Motif, Carlsbad, CA).

Techniques: Binding Assay, Activity Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: NLRC4 inflammasome-mediated production of IL-1? modulates mucosal immunity in the lung against Gram-negative bacterial infection

doi: 10.4049/jimmunol.1200195

Figure Lengend Snippet: A. Human monocyte-derived macrophages (0.5 x106) were transfected with 40 nM NLRC4 siRNA or scrambled siRNA (nonspecific; NS) for 48 h. Cells were then infected with 1 MOI of Kp for 6 h and cytokine/chemokine levels in supernatants were measured by sandwich ELISA. Means ± SE values were obtained from three independent experiments. *P < 0.05 comparing NLRC4 siRNA- with scrambled (control) siRNA-treated cells. B. Human macrophages (0.5 x106) were transfected with siRNA and then infected with 1 MOI of Kp for 6 h and cell lysates were used for the measurement of NF-κB and MAPK activation by western blotting. Representative blots are shown from three blots/experiments with identical results. C. Densitometry was performed from three separate blots using Gel Digitizing Software and normalized to GAPDH (for NF-κB activation) or total p38 (for MAPK activation). Means ± SE values were obtained from three separate experiments. *P < 0.05 comparing NLRC4 siRNA-transfected with control siRNA-transfected macrophages. D. Mouse alveolar macrophages (0.5 X 106 cells/mL) were challenged with 1 MOI of Kp for different time intervals, and culture supernatants were harvested for cytokine/chemokine determination by ELISA. Means ± SE values were obtained from three independent experiments. *P < 0.05 comparing NLRC4−/− with NLRC4+/+ macrophages. E. Mouse alveolar macrophages infected with 1 MOI of Kp were used for the assessment of NF-κB and MAPK activation by western. Representative blots are shown from three blots/experiments with identical results. F. Immunoreactive bands were quantified by densitometry and normalized to GAPDH (for NF-κB activation) or total p38 (for MAPK activation). Data are expressed as means ± SE (n = 3). NS, non-specific

Article Snippet: NF-κB DNA binding assay An ELISA-based NF-κB DNA binding assay in the lungs of saline treated and Kp-infected NLRC4 −/− and NLRC4 +/+ mice was performed to detect the activation of the p65 subunit of NF-κB in the nucleus as per the manufacturer’s protocol (Active Motif, Carlsbad, CA).

Techniques: Derivative Assay, Transfection, Infection, Sandwich ELISA, Activation Assay, Western Blot, Software, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: NLRC4 inflammasome-mediated production of IL-1? modulates mucosal immunity in the lung against Gram-negative bacterial infection

doi: 10.4049/jimmunol.1200195

Figure Lengend Snippet: A. NLRC4 ablation decreases NF-κB DNA binding in lung homogenates following Kp infection. NLRC4+/+ and NLRC4−/− mice were infected with 1 × 103 CFUs of Kp/mouse i.t. and lungs were obtained 24 h and 48 h post-infection. Nuclear fractions obtained from lung homogenates were used for NF-κB binding ELISA. The values are means ± standard errors. Values significantly different between the Kp- and saline-treated groups are indicated by asterisks (P < 0.05; 4–6 mice/group). OD450 nm, optical density at 450 nm. B. NLRC4 deficiency reduces NF-κB activation and expression of ICAM-1 and VCAM-1 in the lung after Kp exposure. NLRC4+/+ and NLRC4−/− mice were infected with 1 × 103 CFUs of Kp/mouse i.t. and lungs were obtained 24 h and 48 h post-infection. Lung homogenates were used for activation of NF-κB and expression of ICAM-1 and VCAM-1 by Western blotting. Representative blot is shown from three blots/experiments with identical results. C. Protein immunoblot bands were quantified by densitometry and normalized to GAPDH. Data are expressed as means ± SE (n = 3). D. NLRC4 ablation reduces MAPK activation in the lung following Kp exposure. NLRC4+/+ and NLRC4−/− mice were infected with 1 × 103 CFUs of Kp/mouse i.t. and lungs were obtained 24 h and 48 h post-infection. Lung homogenates were used to determine activation of MAPKs by western blotting. A representative blot is shown from three blots/experiments with identical results. E. The intensity of immunoreactive bands was quantified by densitometry and normalized to total p38. Data are expressed as means ± SE (*, P < 0.05 between NLRC4+/+ with NLRC4−/− mice; n = 3/group).

Article Snippet: NF-κB DNA binding assay An ELISA-based NF-κB DNA binding assay in the lungs of saline treated and Kp-infected NLRC4 −/− and NLRC4 +/+ mice was performed to detect the activation of the p65 subunit of NF-κB in the nucleus as per the manufacturer’s protocol (Active Motif, Carlsbad, CA).

Techniques: Binding Assay, Infection, Enzyme-linked Immunosorbent Assay, Activation Assay, Expressing, Western Blot

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: NLRC4 inflammasome-mediated production of IL-1? modulates mucosal immunity in the lung against Gram-negative bacterial infection

doi: 10.4049/jimmunol.1200195

Figure Lengend Snippet: A. IL-1R1−/− mice are unable to protect mice and control bacterial growth during acute Kp infection. Mice treated with 1 × 103 CFUs of Kp i.t., and survival was monitored up to 15 days (*, P < 0.05 between NLRC4+/+ with NLRC4−/− mice; n = 20/group). In the other set of experiments, lung homogenates were cultured 24 h or 48 h later. B. Data shown represent mean parenchymal CFUs ± SE (*P < 0.05 comparing NLRC4+/+ with NLRC4−/− mice; n = 4–6/group). C–G. Reduced inflammatory cell recruitment (C–D), and cytokines/chemokines (E–G) in BALF of IL-1R1−/− mice in response to Kp infection. Infiltrating leukocytes from the BALF of IL-1R1−/− and wild-type (C57Bl/6) mice were enumerated on day 1 and 2 after Kp infection. (*, P < 0.05 between NLRC4+/+ with NLRC4−/− mice; n = 4–6/group). H. IL-1R ablation results in reduced NF-κB and MAPK activation as well as expression of ICAM-1 and VCAM-1 in the lung after Kp exposure. IL-1R1+/+ and IL-1R1−/− mice were infected with 1 × 103 CFUs of Kp/mouse i.t. and lungs were obtained 24 h and 48 h post-infection. Lung homogenates were used to assess activation of NF-κB and MAPK as well as expression of ICAM-1 and VCAM-1 by immunoblotting. A representative blot is shown from 3 blots/experiments with identical results. I. Protein immunoblot bands were quantified by densitometry and normalized to GAPDH or total p38. Data are expressed as means ± SE (n = 3). J. Administration of rIL-1β after Kp infection did not rescue neutrophil recruitment in IL-1R1−/− mice. IL-1R1−/− (KO) and IL-1R1/NLRC4−/− (DKO) mice were inoculated i.t. with Kp (1 × 103 CFUs in 50 μL of PBS) and then administered a single dose of recombinant murine IL-1β or vehicle (0.1% BSA). Total neutrophils in BALF (C) were enumerated 48 h after exposure (n = 4–6/group).

Article Snippet: NF-κB DNA binding assay An ELISA-based NF-κB DNA binding assay in the lungs of saline treated and Kp-infected NLRC4 −/− and NLRC4 +/+ mice was performed to detect the activation of the p65 subunit of NF-κB in the nucleus as per the manufacturer’s protocol (Active Motif, Carlsbad, CA).

Techniques: Infection, Cell Culture, Activation Assay, Expressing, Western Blot, Recombinant

Journal: International Immunology

Article Title: Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells

doi: 10.1093/intimm/dxy045

Figure Lengend Snippet: Expression of inflammatory mediators by BMDCs. (A) Effects of goblet cell conditioned media (CjCM) or RA compared to control day 9 untreated group on relative fold expression of Th1 cytokines IL-12 and IFN-γ in cultured BMDCs with or without LPS treatment, measured by RT–PCR. Results expressed as mean ± SEM, n = 4, ***P < 0.005. (B) IL-12 intra-cellular staining of BMDCs detected by flow cytometry (mean ± SEM, n = 3). Untreated (UT; i.e. no LPS) versus LPS treated within group, *P < 0.05; †P < 0.05 comparison between day 9 UT versus CjCM UT. (C) Level of NF-kB p65 phosphorylation in DCs with/without CjCM or RA treatment. Day 9 BMDCs were seeded in 96-well plates treated without or with LPS for 45 min and phospho-p65 (ser536) and total p65 were measured by cell-based enzyme-linked immunosorbent assay. Results expressed as %phospho-p65/total p65 are mean ± SEM of three independent experiments. (D) Effects of CjCM or RA compared to control group on relative fold expression of chemokine receptor CCR7 and ICAM-1 in BMDCs (mean ± SEM, n = 4), ****P < 0.001. UT versus LPS within groups: *P < 0.05, **P < 0.01; between-group comparisons: †P < 0.05, ††P < 0.01, †††P < 0.005.

Article Snippet: NF-κB p65 activation was quantitatively measured by a Fast-activated cell-based ELISA (FACE™) NF-κB p65 Profiler Kit (Active Motif, Carlsbad, CA, USA) that specifically measures phosphorylated and total NF-κB p65.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Cigarette smoke downregulates Nur77 to exacerbate inflammation in chronic obstructive pulmonary disease (COPD)

doi: 10.1371/journal.pone.0229256

Figure Lengend Snippet: Inflammatory responses in lung tissue of WT and Nur77 KO mice exposed to filtered air or CS for two months. Total cell counts ( A ) and photomicrographs (20× objective lens) ( B ) of Diff-Quik-stained cells in BAL fluid from the indicated treatment groups. The following markers were measured in lung tissue homogenates by ELISA: activities of transcription factors (Nur77 [ C ] and NF-κB p65 [ D ]) and the levels of cytokines (TNFα [ E ] and IL-6 [ F ]) and chemokines (MCP-1 [ G ] and KC [ H ]). ( C ) Infrared assay endpoint signal (IR 700 ) represents Nur77 transcriptional activity. ( D ) Spectrophotometric reading value (A 450 ) represents NF-κB p65 activity. ( E - H ) The levels of cytokines and chemokines are shown as protein concentrations (pg) in ml of lung tissue homogenates. Data are expressed as the mean ± SD with n = 5 mice/group; *** P < 0.001, n . s . = non-significant.

Article Snippet: ELISA-based cytokine and chemokine measurements (DTA00D [human tumor necrosis factor-α, TNFα], D8000C [human interleukin 8, IL-8], MTA00B [mouse TNFα], M6000B [mouse interleukin 6, IL-6], MJE00B [mouse monocyte chemoattractant protein-1, MCP-1], and MKC00B [mouse KC]; R&D Systems, Minneapolis, MN) and ELISA-based transcription factor-DNA binding assay (40096 [NF-κB p65]; Active Motif, Carlsbad, CA) were done according to the manufacturer’s instructions and as described previously [ , ].

Techniques: Diff-Quik, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: PLoS ONE

Article Title: Cigarette smoke downregulates Nur77 to exacerbate inflammation in chronic obstructive pulmonary disease (COPD)

doi: 10.1371/journal.pone.0229256

Figure Lengend Snippet: ( A-C ) Nur77 mRNA ( A ), protein ( B ) expression, and Nur77 transcriptional activity ( C ) in H292 cells exposed to filtered air-treated medium (control medium; Air) or CSE for indicated dose and time, determined by real-time PCR ( A ), Western blotting (β-Actin served as a loading control) ( B ), and NurRE binding assay ( C ). For the dose study, cells were exposed to the control medium or CSE for 6 hours ( upper panel ). For the time-course study, cells were treated with the control medium or 10% CSE ( lower panel ). ( D and E ) Nur77 protein expression ( D ) and transcriptional activity ( E ) in control medium-treated or CSE-exposed H292 cells pretreated with Csn-B (10 μM) or Veh, determined by Western blotting (β-Actin served as loading control) ( D ) and NurRE binding assay ( E ). ( F ) The level of Thr-phosphorylated Nur77 in control medium-treated or CSE-exposed H292 cells, by Western blotting. Cells were pretreated with 10 μM of indicated compounds. SB; the p38 MAPK inhibitor SB203580. PD; the ERK inhibitor PD98059. β-Actin served as an input control. ( G ) Levels of inflammatory response markers (NF-κB p65 activity and TNFα and IL-8 production, by ELISA) in control medium-treated or CSE-exposed H292 cells, pretreated with either Csn-B (10 μM) or Veh. ( left panel ) Spectrophotometric reading value (A 450 ) represents NF-κB p65 activity. ( middle and right panels ) The levels of cytokine and chemokine are shown as protein concentrations (pg) in ml of cell culture medium. Data are expressed as the mean ± SD with n = 3; *** P < 0.001, n . s . = non-significant.

Article Snippet: ELISA-based cytokine and chemokine measurements (DTA00D [human tumor necrosis factor-α, TNFα], D8000C [human interleukin 8, IL-8], MTA00B [mouse TNFα], M6000B [mouse interleukin 6, IL-6], MJE00B [mouse monocyte chemoattractant protein-1, MCP-1], and MKC00B [mouse KC]; R&D Systems, Minneapolis, MN) and ELISA-based transcription factor-DNA binding assay (40096 [NF-κB p65]; Active Motif, Carlsbad, CA) were done according to the manufacturer’s instructions and as described previously [ , ].

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a–f Primary human midbrain astrocyte cultures were treated for 24 h with PFFs or PBS control solution. Cultures were pretreated (30 min) with inhibitors of NF-κB (BAY), JAK/STAT (PYR), or AP1 (SR) signaling prior to the addition of PFFs. Levels of indicated A1-associated transcripts ( a ), A2-associated transcripts ( b ), and inflammatory chemokines ( c–e ) were measured using qRT-PCR. f Levels of CXCL10 protein in culture supernatants were measured via ELISA. Data in ( a ) and ( b ) were normalized and z-transformed summaries of the qPCR data that appears in Supplementary Figs. and , represented via heatmaps. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates for all experiments.

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a–d qRT-PCR analysis of indicated genes in human midbrain astrocyte cultures treated following 24 h PFF treatment ± cotreatment with JSH-23. e Confocal microscopy of NF-κB component p65 (red) and nuclei (DAPI, blue) in human midbrain astrocytes following 2 h PFF treatment ± cotreatment with BAY. Scale bars = 20 μm. f Colocalization of red and blue signal in ( e ) is quantified as Fisher’s Z transformed index of correlation. g Nuclear fractions were isolated from human midbrain astrocyte cultures following 2 h PFF treatment ± cotreatment with BAY. Relative levels of p65 in nuclear fractions were quantified using ELISA. h , i Secondary analysis of microarray profiling of ( h ) rat astrocytes treated with conditioned neuronal medium containing LacZ or α-synuclein (GSE11574) or i human postmortem substantia nigra samples from patients with PD or healthy controls (GSE26927). Z scores were calculated for genes under the GO term 0038061 and those exhibiting significantly differential expression between groups (corrected p value < 0.05) are displayed in the respective heatmaps. j–m qRT-PCR analysis of indicated genes in human midbrain astrocyte cultures treated following 24 h PFF treatment ± cotreatment with indicated inhibitors. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates for all experiments.

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Quantitative RT-PCR, Confocal Microscopy, Transformation Assay, Isolation, Enzyme-linked Immunosorbent Assay, Microarray, Expressing

Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a–e Primary human midbrain astrocyte cultures were treated for 24 h with PFFs or PBS control solution. Cultures were pretreated (30 min) with inhibitors of NF-κB (BAY), JAK/STAT (PYR), or AP1 (SR) signaling prior to the addition of PFFs. a–d Levels of indicated phagocytosis-associated transcripts were measured using qRT-PCR. e Twenty-four hours following PFF or PBS treatment, relative rates of phagocytosis were assessed by measuring uptake of fluorescently labeled zymosan. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates for all experiments.

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Quantitative RT-PCR, Labeling

Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a Confocal microscopy of NF-κB component p65 (red) and nuclei (DAPI, blue) in human midbrain astrocytes following 2 h PFF treatment ± cotreatment with indicated inhibitors. Scale bars = 50 μm. b Colocalization of red and blue signal in ( a ) is quantified as Fisher’s Z transformed index of correlation. c Nuclear fractions were isolated from human midbrain astrocyte cultures following 2 h PFF treatment ± cotreatment with indicated inhibitors. Relative levels of p65 in nuclear fractions were quantified using ELISA. d–j Primary human midbrain astrocyte cultures were treated with PFFs or PBS control solution. Cultures were pretreated (30 min) with inhibitors of RIPK3 (GSK872) or NF-κB (BAY) signaling prior to the addition of PFFs. Levels of indicated transcripts were measured at indicated time points using qRT-PCR. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates in ( a–c ). n = 3 independent replicates in ( d–j ).

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Confocal Microscopy, Transformation Assay, Isolation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

doi: 10.1038/s41419-021-04049-0

Figure Lengend Snippet: a Primary human midbrain astrocytes were treated with RIPK inhibitors and/or PFFs, as indicated. After 24 h, an astrocyte conditioned medium (ACM) was applied (1:1) to differentiated SH-SY5Y cultures for 24 h followed by endpoint analyses. b Viability of SH-SY5Y cells following treatment with ACM derived from astrocyte cultures treated with the indicated inhibitors was measured via ATP-luciferase assay (Cell Titer Glo). c , d Primary human midbrain astrocyte cultures were treated with PFFs or PBS control solution. Cultures were pretreated (30 min) with inhibitors of RIPK3 (GSK872) or NF-κB (JSH-23) signaling prior to the addition of PFFs. Following this, CSFE-labeled neuronal debris was added for 24 h. Uptake was measured via flow cytometric analysis of CSFE signal in astrocytes. Data were represented as histograms ( c ) and quantified using mean geometric fluorescence intensity (GMFI) values ( d ). e Secondary analysis of microarray profiling of human postmortem substantia nigra samples from patients with PD or healthy controls (GSE26927). Z -scores and corrected p values were calculated for necroptosis pathway components indicated and displayed via heatmap. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means. n = 6 independent replicates in ( b ). n = 3 independent replicates in ( c , d ).

Article Snippet: Equal amounts of protein were then processed through an ELISA-based kit for detecting p65 (NF-κB p65 Transcription Factor Assay Kit, Abcam, Cambridge, MA, #ab133112).

Techniques: Derivative Assay, Luciferase, Labeling, Fluorescence, Microarray

Journal: Molecular Medicine Reports

Article Title: γ-tocotrienol attenuates TNF-α-induced changes in secretion and gene expression of MCP-1, IL-6 and adiponectin in 3T3-L1 adipocytes

doi: 10.3892/mmr.2012.770

Figure Lengend Snippet: Effect of γ-tocotrienol on NF-κB activity in TNF-α-treated 3T3-L1 adipocytes. 3T3-L1 adipocytes were pre-treated with various concentrations of γ-tocotrienol (0.024–2.4 μM) for 6 h and then exposed to 10 ng/ml TNF-α for 24 h. The nuclear fraction was obtained and NF-κB (p65) levels were assayed by ELISA, as described in Materials and methods. Values are expressed relative to the untreated group. Data are expressed as the means ± SEM (n=5). ## P<0.01 vs. the untreated group; ** P<0.01 vs. the TNF-α only-treated group.

Article Snippet: To quantify NF-κB activity, nuclear extracts were prepared using Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) and analyzed with a sensitive ELISA-based kit (PathScan Total NF-κB p65 Sandwich ELISA kit; Cell Signaling Technology) to quantify NF-κB activity, according to the manufacturer's instructions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: The IκB Function of NF-κB2 p100 Controls Stimulated Osteoclastogenesis

doi: 10.1084/jem.20030116

Figure Lengend Snippet: NF-κB does not accumulate in the nucleus of NIK −/− preOCs. (A) PreOCs, generated by treating Mφs with RANKL for 48 h followed by a 3 h starvation, were stimulated with RANKL for the indicated times, and cytoplasmic (top) and nuclear (bottom) fractions were isolated and analyzed by immunoblot for IκBα and RelA, respectively. Although IκBα gets degraded in both WT and NIK −/− cultures, RelA does not accumulate in the nucleus in the absence of NIK. (B) Nuclear extracts prepared as in A were analyzed for NF-κB DNA binding activity by EMSA. Whereas WT preOC cultures have detectable NF-κB activity at baseline and further induction by RANKL, NIK −/− preOC cultures show little binding activity with or without RANKL. (C) RelA-specific NF-κB DNA binding activity was assessed by ELISA (EZ Detect kit; Pierce Chemical Co.) using the same preOC nuclear extracts as in A. In this assay, NF-κB–specific oligos are bound to a plate. Nuclear extract is then applied followed by anti-p65 (RelA) antibody and an HRP-conjugated secondary. The assay is developed with chemiluminescent reagents and read on a luminometer/plate reader. Each sample was run in triplicate. Unmutated (UnMu) and mutant (Mu) soluble oligos were added to the WT 30-min sample to confirm specificity. The results of this assay mirror the EMSA in B and confirm lower baseline NF-κB activity and lack of RANKL-mediated induction in NIK −/− preOCs. Binding activity is abolished by addition of the unmutated oligo and unaffected by the mutant oligo (last two bars).

Article Snippet: For RelA supershift, 2 μg polyclonal anti-RelA (Santa Cruz Biotechnology, Inc.) was added after formation of DNA–protein complexes at room temperature for 15 min. An ELISA-based NF-κB assay, the EZDetect p65 ELISA (Pierce Chemical Co.) was performed according to manufacturer instructions, using 2 μg nuclear extract per well, performed in triplicate.

Techniques: Generated, Isolation, Western Blot, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis