Journal: Cell stem cell

Article Title: PGC-1α Controls Skeletal Stem Cell Fate and Bone-Fat Balance in Osteoporosis and Skeletal Aging by Inducing TAZ

doi: 10.1016/j.stem.2018.06.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-NFkB p65 NLS specific , Rockland , Cat#600-401-271 RRID: AB_2269511.

Techniques: Control, Recombinant, Reverse Transcription, Transfection, Red Blood Cell Lysis, Virus, Staining, Diagnostic Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Software, Microscopy, Imaging

Journal: Bioengineered

Article Title: C1q/tumor necrosis factor-related protein-3 (CTRP3) activated by forkhead box O4 (FOXO4) down-regulation protects retinal pericytes against high glucose-induced oxidative damage through nuclear factor erythroid 2-related factor 2 (Nrf2)/Nuclear factor-kappaB (NF-κB) signaling

doi: 10.1080/21655979.2022.2031413

Figure Lengend Snippet: CTRP3 inactivated by FOXO4 modulates the Nrf2/NF-κB signaling pathway. The protein levels of Nrf2, p-NF-κB p65 and NF-κB p65 were analyzed by Western blot. ***P < 0.001 vs. MA. ### P < 0.001 vs. HG+Ov-NC. $$$ P < 0.001 vs. HG+Ov-CTRP3+ Ov-NC. CTRP3, C1q/tumor necrosis factor-related protein-3. HG, high glucose. MA, mannitol. FOXO4, forkhead box O4. Nrf2, nuclear factor erythroid 2-related factor 2. p-NF-κB p65, phosphorylated-Nuclear factor-kappaB p65. NF-κB p65, Nuclear factor-kappaB p65.

Article Snippet: The membranes were impeded by 5% nonfat milk and incubated with the following primary antibodies: anti-CTRP3 (Abcam, ab36870), anti-B cell lymphoma-2 (Bcl-2) (Abcam, 1:1000, ab32124), anti-BCL-2 associated X (Bax) (Abcam, 1:1000, ab32503), anti-cleaved caspase 3 (Abcam, 1:500, ab32042), anti-caspase 3 (Abcam, 1:5000, ab32351), anti-cleaved Poly (ADP-ribose) polymerase (PARP) (Abcam, 1:1000, ab32064), PARP (Abcam, 1:1000, ab191217), anti-FOXO4 (Abcam, 1:1000, ab128908), anti-Nrf2 (Abcam, 1:1000, ab62352), anti- phosphorylated (p-)NF-κB p65 (Abcam, 1:1000, ab76302), anti-NF-κB p65 (Abcam, 1:1000, ab32536) and anti-GAPDH (Abcam, 1:2500, ab9485) at 4°C overnight.

Techniques: Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Anti-phospho-p65 Ser276 (Cell Signaling no. 3037) detects atypical (with MWs of 80 and 130 kDa), inducible immunoreactivities in different cell types, induced with different NF- κ B-activating agents. Cells were stimulated for increasing times (L929sA, A549, L363) or for 30 min (Raw264.7, C2C12, 1321N1) with TNF- α (2000 IU/mL), LPS (1 μ g/mL), PMA (10 ng/mL), forskolin (forsk, 10 μ M) or isoproterenol (iso, 10 μ M). For blocking experiments, lysates were loaded on one gel in duplicate and after transfer, blots were cut in half. Immunodetection was performed in parallel, using Cell Signaling no. 3037 or Cell Signaling no. 3037 preincubated for at least 30 minutes with double volume of blocking peptide (BLOCK). Arrow 1 indicates an immunoreactive band with an MW of 130 kDa; arrow 2 indicates an immunoreactive band of 80 kDa.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Blocking Assay, Immunodetection

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The Cell Signaling no. 3037 anti-P-p65 Ser276-immunoreactive band persists in cells where p65 is silenced via an siRNA approach. 1321N1 cells were transiently transfected with control or p65 siRNA as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M), or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1). 130 and 80 kDa immunoreactivities are indicated by arrows. Western Blot to detect anti-P-p65Ser536 (65 kDa arrow) was performed on the same lysates (B1). Blots A1 and B1 were extensively washed and reprobed with anti-p65 to investigate efficiency of p65 knock-down. Anti-p65 detected bands are marked with arrows (A2, B2). Finally, blots were reprobed with anti-tubulin to check loading efficiency. Anti-tubulin-detected bands are marked with arrows (A3, B3).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Transfection, Control, Western Blot, Knockdown

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Four independent anti-P-p65 Ser276 antibodies detect inducible bands, with MWs of 80 and 130 kDa, that do not disappear upon p65 knock-down, but are inhibited when PKAc α is silenced. 1321N1 cells were transiently transfected with control, p65 or PKAc α siRNA, as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M) or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using SAB no. 11011 (A1), Rockland no. 100-401-264 (B1), Cell Signaling no. 3037 (B3), or a homemade anti-P-p65 Ser276 antibody (C1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2, resp.). Blots were extensively washed and reprobed with a mixture of anti-p65 and anti-PKAc α to investigate knock-down efficiency. Anti-p65-detected bands are marked by the upper arrow, anti-PKAc α -reactive bands are marked by the lower arrow (A2, B2, C2). Tubulin loading controls are added as Supplementary Figure 1.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Knockdown, Transfection, Control, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Presence of anti-P-p65 Ser276-reactive bands in p65 deficient MEF cells. p65 knock-out MEF cells (−/−), or p65 −/− MEF cells reconstituted with wild type p65 (p65 wt) or p65 with Ser276 mutated to alanine (p65 S/A), were induced with forskolin (forsk, 10 μ M) for 0, 30, and 60 min. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1) or SAB no. 11011 (B1). Blots were extensively washed and reprobed with anti-p65 to confirm p65 absence in −/− MEFs, and p65 presence in wt and S/A reconstituted MEFs (A2 and B2, arrows indicate anti-p65 detected bands).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Knock-Out, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The detected 130 kDa and 80 kDa immunoreactivities are not NF- κ B p105/p50 or c-Rel. 1321N1 cells were transiently transfected with control, p105/p50 or c-Rel siRNA as described in . Cells were induced for 30 min with TNF- α (2000 IU/mL) or iso (10 μ M). Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1, B1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2 resp.). Because of the overlap of the 130 kDa and the p105 immunoreactive bands, blot A was first stripped and reprobed with anti-p105/p50 (A2). p105 immunoreactivity is indicated by the upper arrow; p50 immunoreactivity is indicated by the lower arrow. Blot B was extensively washed and reprobed (without stripping) with anti-c-Rel (B2). c-Rel immunoreactivity is indicated by an arrow. Tubulin was used as a loading control (A3, B3).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Transfection, Control, Western Blot, Stripping Membranes

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: In vitro recognition of the phosphorylated Serine 276 residue of p65 by Cell Signaling no. 3037. Recombinant wt p65-GST or SC mutant p65-GST fusion proteins were in vitro phosphorylated using recombinant MSK-1. 250 ng of the recombinant proteins were spotted on a nitrocellulose membrane and subjected to Western Blotting with Ser276 phospho-specific p65 antibody, either preincubated with 10 μ g/mL phosphorylated peptide that was used to generate the antibody (upper panel) or not. 1 μ g of recombinant protein was separated by SDS-PAGE after the in vitro kinase assays performed either in the presence of active MSK-1 and/or 10 μ M H89 (lower panel) or not.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: In Vitro, Residue, Recombinant, Mutagenesis, Membrane, Western Blot, SDS Page

Journal: Oncogene

Article Title: Cyclin E and Bcl-xL cooperatively induce cell cycle progression in c-Rel-/- B cells.

doi: 10.1038/sj.onc.1206917

Figure Lengend Snippet: Figure 6 c-Rel regulates E2F3a expression in B cells. (a) Resting splenic B cells from c-rel þ / þ; bcl-xLTg (WT Tg) and c-rel/; bcl- xLTg (KO Tg) B cells were cultured for 36 h in the presence of 10 mg/ml anti-IgM and assayed by RT–PCR using primers specific for E2F3a. (b) Approximately 1 kb of mouse e2f3a promoter sequence is shown with two putative NF-kB/Rel-binding sites indicated by red boxes, as determined by Transfac database analysis. Site I corresponds to 864 bp upstream of the ATG translational start site ( þ 1) and site II is located 214 bp upstream. (c) Cells were cultured as described in (b) and assayed for NF-kB/Rel-binding activity by gel-shift assay using a radi- olabeled probe containing the 864 kB site and antibodies specific for p50, p65, and c-Rel to supershift complexes. The inducible complex (labeled A) is supershifted by p50, c-Rel, and p65 antibodies in wild-type B cells stimulated with 10 mg/ml anti-IgM, but a mutated site (864 kBMUT) prevents the binding of these complexes. (Supershifted complexes do not visibly migrate, but appear lighter than nonshifted control bands instead.) An additional site (214 kB) does not appear to bind any NF-kB/ Rel complexes either. Complex B represents nonspecific binding activity that serves to show equal loading of lanes. (d) CH31 B cells stimulated with 10 mg/ml anti-CD40 also exhibit c-Rel and p50 binding activity (labeled A) at the 864 kB site of the e2f3a promoter but a mutant 864 kB site and the 214 kB site do not bind. (e) WEHI 231 B cells stimulated with 10 mg/ml anti-CD40 show NF-kB/Rel binding activity (labeled A) while the mutant 864 kB site and 214 kB site do not exhibit any NF-kB/Rel- binding activity. (f) A total of 1 105 NIH 3T3 cells were transfected with 0.5 mg of firefly luciferase constructs containing a 2 kb e2f3a promoter (E2F3a-luc), a mutant 2 kb promoter (MUT- luc) bearing a mutation at the 864 kB site, or a positive control Igk chain promoter (Igk-luc), and cotransfected with either 0.5 mg of p50, c-Rel, or empty expression vector (pcDNA3). Cells were also cotransfected with a Renilla luciferase internal control plasmid containing a thymidine kinase promoter (tk-pRL) to drive constitutive Renilla luciferase activity for normalization of data. At 48 h, cells were harvested and assayed for firefly and Renilla luciferase activity. All experiments were carried out in triplicate and repeated in three separate experiments. A representative set of data is shown

Article Snippet: Supershift analysis was performed using 5–7mg anti-p130 (sc-317 ), anti-p107 (sc-317 ), anti-cRel (sc-071) Santa Cruz Biotechnology, anti-p50 from previous studies (Liou et al., 1994), and anti-p65 (RelA; 100–4165) from Rockland.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Binding Assay, Activity Assay, Gel Shift, Labeling, Control, Mutagenesis, Transfection, Luciferase, Construct, Positive Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro and In Vivo Validation of Endothelium-Derived Potential Therapeutics for Myocardial Ischemia/Reperfusion Injury Identified by an AI-Enhanced Single-Cell and Virtual-Cell Paradigm

doi: 10.3390/ijms27062743

Figure Lengend Snippet: Andrographolide inhibits the S100A8 downstream IL-17 signaling pathway and reduces the inflammatory response of H/R-induced CMECs. ( A , B ) The expressions of IL-17A, IL-17RA, Act1, and NF-κB proteins were determined by Western blot ( n = 3). ( C – E ) The levels of IL-1β, IL-6, and TNF-α were determined through ELISA. ( F – H ) The expressions of IL-17A, IL-17RA and Act1 proteins were determined by Western blot in co-transfection with the overexpression S100A8 plasmid ( n = 3). Data are presented as mean ± SD ( n = 6). “Con or C” stands for the control group, “H/R” or “H” indicates the H/R group, and “H + AG or H + A” means the H/R group treated with AG. “H + A + S” means the H/R group treated with AG and overexpression of S100A8. ** p < 0.01 compared to the Con group; # p < 0.05, ## p < 0.01 compared to the H/R or H group, @ p < 0.05, @@ p < 0.01 compared to the H + A + S group.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skimmed milk before being incubated overnight at 4 °C with the following primary antibodies: S100A8 (A15315, Abclonal, Wuhan, China), IL-17A (A12454, Abclonal, Wuhan, China), IL-17RA (A10052, Abclonal, Wuhan, China), Act1 (A6776, Abclonal, Wuhan, China), p-NF-κB p65 (1:500, AP0124, Abclonal, Wuhan, China), NF-κB p65 (1:500, A00284-1, Boster, Wuhan, China), β-actin (1:2000, TA-09, Zhongshan Golden Bridge, Beijing, China), and GAPDH (1:2000, TA-08, Zhongshan Golden Bridge, Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cotransfection, Over Expression, Plasmid Preparation, Control

Journal: PLoS ONE

Article Title: Downregulation of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in a co-culture system with human stimulated X-linked CGD neutrophils

doi: 10.1371/journal.pone.0230665

Figure Lengend Snippet: (A) Total protein expression of NFκb p65 and phosphorylated at Ser536 in HUVECs in the neutrophil-containing transwell system compared with GAPDH expression. (B) Quantification of total NFκb p65. (C) Quantification of phosphorylation at Ser536 position compared with total NFκb p65.

Article Snippet: For immunodetection, membranes were blocked in 5% BSA in TBS with 0.1% Tween-20 (TBST) and incubated in TBST with eNOS and phosphorylated-eNOS(Ser1177) antibodies (Cell life signaling, Danvers, MA, USA) (1:100 dilution), anti-EDN1 antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:500), anti- NFκB (p65) antibody, and anti-phosphorylated NFκB (p65) at Ser536 position (St. John’s Laboratory, London, UK) (1:500), and anti-GAPDH antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:1000) with chemiluminescent detection.

Techniques: Expressing, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells.

doi: 10.1074/jbc.270.52.31315

Figure Lengend Snippet: FIG. 4. Syntheses of double stranded oligonucleotides as probes for mouse cyclooxygenase-2 gene. g1, 2409 to 2385 bp including the putative NFkB binding site indicated by a solid box; g2, GA of the NFkB was changed to CC; gA, 2155 to 2121 bp including AP2 and NF-IL6; gB, GC of AP2 was changed to TT; gC, TTGCG of the NF-IL6 was changed to CCGCT.

Article Snippet: Materials—The materials used in this work were purchased as follows: [1-14C]arachidonic acid (2.1 GBq/mmol) and 35S-labeled anti-rabbit IgG whole antibody (74 TBq/mmol) from Amersham International (Bucks, United Kingdom); Immobilon-P transfer membrane from Millipore (Bedford, MA); [g-32P]ATP (220 TBq/mmol) from DuPont NEN; fetal bovine serum and newborn bovine serum from Irvine Scientific (Santa Ana, CA); penicillin G and phenylmethylsulfonyl fluoride from Sigma; a-modified Eagle’s minimum essential medium (a-MEM), recombinant mouse TNFa, and lipofectAMINE from Life Technologies, Inc. (Gaithersburg, MD); ISOGEN from Nippon Gene (Tokyo, Japan); EMBL3 mouse genomic library from Clonetech (Palo Alto, CA); Pica Gene luciferase assay system from Toyo Ink (Tokyo, Japan); poly(dIdC)-poly(dI-dC) from Pharmacia (Uppsala, Sweden); anti-AP2, anti-C/ EBPb (NF-IL6), and anti-NFkB p50 antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-NFkB p65 antibody from Serotec (Oxford, United Kingdom); and purified NFkB p50 protein from Promega (Madison, WI).

Techniques: Binding Assay

Journal: The Journal of biological chemistry

Article Title: Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells.

doi: 10.1074/jbc.270.52.31315

Figure Lengend Snippet: FIG. 5. Electrophoretic mobility shift assay targeting the NFkB site. Probe g1 was incubated for 0.5 h with the nuclear extracts (2.3 mg protein) of MC3T3-E1 cells treated with 0 ng/ml (lane 3), 0.2 ng/ml (lane 4), 2 ng/ml (lane 5), and 20 ng/ml (lanes 6 and 9) TNFa. The nuclear extracts before the addition of TNFa were also incubated with g1 (lane 2). Anti-NFkB p50 (lane 10) or anti-NFkB p65 (lane 11) anti- body was incubated together with the nuclear extracts from the cells stimulated with 20 ng/ml TNFa. Purified NFkB p50 protein was incu- bated with probe g1 (lane 7). Probe g2 with a mutation for NFkB was incubated with the nuclear extracts from the cells stimulated with 20 ng/ml TNFa (lanes 13 and 14). Anti-NFkB p50 antibody was also present (lane 14). Lanes 1, 8, and 12 were control runs without the nuclear extracts. Shifted bands indicated by a, b, and g are described in the text.

Article Snippet: Materials—The materials used in this work were purchased as follows: [1-14C]arachidonic acid (2.1 GBq/mmol) and 35S-labeled anti-rabbit IgG whole antibody (74 TBq/mmol) from Amersham International (Bucks, United Kingdom); Immobilon-P transfer membrane from Millipore (Bedford, MA); [g-32P]ATP (220 TBq/mmol) from DuPont NEN; fetal bovine serum and newborn bovine serum from Irvine Scientific (Santa Ana, CA); penicillin G and phenylmethylsulfonyl fluoride from Sigma; a-modified Eagle’s minimum essential medium (a-MEM), recombinant mouse TNFa, and lipofectAMINE from Life Technologies, Inc. (Gaithersburg, MD); ISOGEN from Nippon Gene (Tokyo, Japan); EMBL3 mouse genomic library from Clonetech (Palo Alto, CA); Pica Gene luciferase assay system from Toyo Ink (Tokyo, Japan); poly(dIdC)-poly(dI-dC) from Pharmacia (Uppsala, Sweden); anti-AP2, anti-C/ EBPb (NF-IL6), and anti-NFkB p50 antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-NFkB p65 antibody from Serotec (Oxford, United Kingdom); and purified NFkB p50 protein from Promega (Madison, WI).

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Purification, Mutagenesis, Control

Journal: The Journal of biological chemistry

Article Title: Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells.

doi: 10.1074/jbc.270.52.31315

Figure Lengend Snippet: FIG. 6. Electrophoretic mobility shift assay targeting AP2 and NF-IL6 sites. MC3T3-E1 cells were incubated for 1 h with various concentrations of TNFa. The nuclear extracts (2.3 mg of protein) from the cells stimulated with 0 ng/ml (lane 3), 0.2 ng/ml (lane 4), 2 ng/ml (lane 5), and 20 ng/ml (lanes 6–9, 11–14, and 16–19) TNFa were incubated with probe gA (wild type, lanes 3–9), gB (mutation for AP2, lanes 11–14), or gC (mutation for NF-IL6, lanes 16–19). The nuclear extracts before the addition of TNFa was also incubated with gA (lane 2). Anti-AP2 (lanes 7, 12 and 17), anti-NF-IL6 (lanes 8, 13, and 18), or anti-NFkB p50 (lanes 9, 14, and 19) antibody was incubated together with the nuclear extracts. Lanes 1, 10, and 15 were for incubations without the nuclear extracts. Shifted bands indicated by a-g are de- scribed in the text.

Article Snippet: Materials—The materials used in this work were purchased as follows: [1-14C]arachidonic acid (2.1 GBq/mmol) and 35S-labeled anti-rabbit IgG whole antibody (74 TBq/mmol) from Amersham International (Bucks, United Kingdom); Immobilon-P transfer membrane from Millipore (Bedford, MA); [g-32P]ATP (220 TBq/mmol) from DuPont NEN; fetal bovine serum and newborn bovine serum from Irvine Scientific (Santa Ana, CA); penicillin G and phenylmethylsulfonyl fluoride from Sigma; a-modified Eagle’s minimum essential medium (a-MEM), recombinant mouse TNFa, and lipofectAMINE from Life Technologies, Inc. (Gaithersburg, MD); ISOGEN from Nippon Gene (Tokyo, Japan); EMBL3 mouse genomic library from Clonetech (Palo Alto, CA); Pica Gene luciferase assay system from Toyo Ink (Tokyo, Japan); poly(dIdC)-poly(dI-dC) from Pharmacia (Uppsala, Sweden); anti-AP2, anti-C/ EBPb (NF-IL6), and anti-NFkB p50 antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-NFkB p65 antibody from Serotec (Oxford, United Kingdom); and purified NFkB p50 protein from Promega (Madison, WI).

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Mutagenesis

Journal: The Journal of biological chemistry

Article Title: Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells.

doi: 10.1074/jbc.270.52.31315

Figure Lengend Snippet: FIG. 7. Mutations of NFkB and NF-IL6 motifs. A mixture of each luciferase plasmid which is wild or mutated (cross indicates point mu- tation of wild plasmid) (2.3 mg) and b-galactosidase plasmid (1.2 mg) was transfected into MC3T3-E1 cells (1.4 3 105 cells/60-mm dish) by the lipofection method. The cells were cultured to confluency for 3 days, and incubated for 12 h with (closed column) or without (slashed column) 20 ng/ml TNFa. The luciferase activity was assayed, and normalized with the b-galactosidase activity. Data are means 6 S.E. of triplicate determinations.

Article Snippet: Materials—The materials used in this work were purchased as follows: [1-14C]arachidonic acid (2.1 GBq/mmol) and 35S-labeled anti-rabbit IgG whole antibody (74 TBq/mmol) from Amersham International (Bucks, United Kingdom); Immobilon-P transfer membrane from Millipore (Bedford, MA); [g-32P]ATP (220 TBq/mmol) from DuPont NEN; fetal bovine serum and newborn bovine serum from Irvine Scientific (Santa Ana, CA); penicillin G and phenylmethylsulfonyl fluoride from Sigma; a-modified Eagle’s minimum essential medium (a-MEM), recombinant mouse TNFa, and lipofectAMINE from Life Technologies, Inc. (Gaithersburg, MD); ISOGEN from Nippon Gene (Tokyo, Japan); EMBL3 mouse genomic library from Clonetech (Palo Alto, CA); Pica Gene luciferase assay system from Toyo Ink (Tokyo, Japan); poly(dIdC)-poly(dI-dC) from Pharmacia (Uppsala, Sweden); anti-AP2, anti-C/ EBPb (NF-IL6), and anti-NFkB p50 antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-NFkB p65 antibody from Serotec (Oxford, United Kingdom); and purified NFkB p50 protein from Promega (Madison, WI).

Techniques: Luciferase, Plasmid Preparation, Transfection, Cell Culture, Incubation, Activity Assay