Journal: Mediators of Inflammation

Article Title: Inhibitory Effect of Inflexinol on Nitric Oxide Generation and iNOS Expression via Inhibition of NF- κ B Activation

doi: 10.1155/2007/93148

Figure Lengend Snippet: Effect of inflexinol on LPS-induced NF- κ B activity and degradation of I κ B in RAW 264.7 cells (a) and astrocytes (b). The cell treated with 1 μ g/mL of LPS only or LPS plus diffent concentrations (1, 5, 10 μ M) of inflexinol at 37°C for 1 hour. Equal amounts of total protein (40 μ g) were subjected to 10% SDS-PAGE. Nuclear translocation of p50 and p65, and degradation of I κ B were detected by Western blotting using specific antibodies. β -actin protein was used here as an internal control. Similar results were obtained from at least three different sets of experiment.

Article Snippet: Rabbit polyclonal antibodies against iNOS and COX-2 (1 : 1000) (Cayman Chemical, Ann Arbor, Mich, USA), and rabbit polyclonal antibodies against p65 and I κ B α (1 : 500), and mouse monoclonal antibody against p50 (1 : 500) (Santa Cruz Biotechnology Inc. Santa Cruz, Calif, USA) were used in study.

Techniques: Activity Assay, SDS Page, Translocation Assay, Western Blot, Control

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: IκBβ is specifically regulated by p65 in fibroblasts. (A) Whole-cell extracts (WCEs) were prepared from immortalized MEFs either wild type or null for the p65 subunit of NF-κB, and the levels of the indicated proteins were analyzed by Western blotting. (B) Western blot assays to probe for IκBα and IκBβ in MEFs either wild type or null for various components in the NF-κB signaling pathway. Genotypes for all cell lines were confirmed by PCR analysis. (C) WCEs were prepared from vector or IκBα-SR C2C12 myoblasts, and Western blot assays were performed to probe for IκB proteins. (D) Primary MEFs were isolated from E13.5 embryos and genotyped for p65 by PCR. WCEs were prepared from p65 wild-type, heterozygous, and null MEFs, and Western blot assays were performed to probe for p65, IκBα, and IκBβ. α-Tubulin was used as a loading control. (E) Quantitation of IκB proteins from two Western blot assays performed with WCEs obtained from two independent litters as described for panel D. Levels of IκB proteins were normalized to α-tubulin and compared to the expression of IκB proteins obtained from wild-type cells, which was set to a value of 100%.

Article Snippet: Briefly, p65 +/− TNF -α +/+ mice were crossed to p65 +/+ TNF -α −− mice (Jackson Laboratories, Bar Harbor, Maine).

Techniques: Western Blot, Plasmid Preparation, Isolation, Control, Quantitation Assay, Expressing

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: p65 regulation of IκBβ is not limited to fibroblasts. (A) Histological hematoxylin-and-eosin (H&E) and immunohistochemical staining of p65 and IκBβ from longitudinal sections of p65+/+ (a, b, c) and p65−/− (d, e, f) embryos (×1 magnification). (B) Immunohistochemical staining of IκBβ of liver (a, d), lung (b, e), and brain (c, f) tissues from p65+/+ and p65−/− embryos (×4 magnification). (C) Primary fetal liver cells were prepared from E13.5 embryos, and after genotypes were confirmed, Western blot assays were performed to probe for p65, IκBα, and IκBβ. α-Tubulin was used as a loading control. (D) p65 and IκBβ immunohistochemical staining of heart tissue from p65+/+ and p65−/− embryos (×4 magnification). (E) p65 and IκBβ immunohistochemical staining of forelimbs from p65+/+ and p65−/− embryos. To confirm skeletal muscle staining, serial sections of forelimbs were separately stained for myosin heavy chain (MyHC; arrowheads denote muscle tracks; ×4 magnification).

Article Snippet: Briefly, p65 +/− TNF -α +/+ mice were crossed to p65 +/+ TNF -α −− mice (Jackson Laboratories, Bar Harbor, Maine).

Techniques: Immunohistochemical staining, Staining, Western Blot, Control

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: p65 regulates various forms of IκBβ in postnatal development in a tissue-specific manner. p65+/− TNF-α−/− mice were bred to generate p65+/+ TNF-α−/− and p65−/− TNF-α−/− progeny. (A) At 4 weeks of age, mice were sacrificed and tissue homogenates were prepared (skmc, skeletal muscle). Western blot assays were then performed to probe for p65, IκBα, IκBβ, and α-tubulin. Each Western blot assay is representative of a total of four different blot assays derived from two sets of littermates. (B) Western blot assay to probe for IκBβ in p65+/+ TNF-α−/− brain, skin, skeletal muscle, and heart tissues. Arrows denote IκBβ forms I through IV. (C) Similar extracts as in panel A were either left untreated or treated with phosphatase (PPase) enzyme, and Western blot assay was performed to probe for IκBβ. Arrows denote phosphorylated forms of IκBβ, and asterisks denote the shifted (dephosphorylated) forms of IκBβ. (D) The N-terminal IκBβ antibody (N20) was used in a Western analysis to verify IκBβ forms in p65+/+ TNF-α−/− tissues.

Article Snippet: Briefly, p65 +/− TNF -α +/+ mice were crossed to p65 +/+ TNF -α −− mice (Jackson Laboratories, Bar Harbor, Maine).

Techniques: Western Blot, Derivative Assay

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: IκBβ downregulation in p65−/− MEFs is regulated by the proteasome independent of classical IKK signaling. (A) MEFs wild type and null for p65 were treated with TNF-α, and at indicated time points a real-time PCR was used to measure IκBα and IκBβ RNAs. (B) p65+/+ and p65−/− MEFs were incubated for 2 h in methionine- and cysteine-free DMEM and subsequently labeled with a [35S]methionine/cysteine mix for an additional hour. Whole-cell extracts were prepared, and IκBβ was immunoprecipitated either with IgG (control) or with an IκBβ-specific antibody. Complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by exposing dried gels to film for up to 3 days (arrow denotes IκBβ). (C) p65+/+ and p65−/− MEFs were treated with cycloheximide (CHX; 10 μg/ml), and at indicated times whole-cell extracts were prepared and Western blot assays performed to probe for IκBβ. Levels of IκBβ were quantitated from an average of three Western blot assays. (D) Similar conditions were used to treat p65+/+ and p65−/− MEFs with MG-132, ALLN, and lactacysteine (all at 10 μM). (E) Kinase assays (KA) were performed with IKKγ immunoprecipitates from p65+/+ and p65−/− MEFs incubated with glutathione S-transferase-IκBα as a substrate. Whole-cell extracts from p65+/+ and p65−/− MEFs used in kinase assay were analyzed by Western bloting to probe for IKKα and p65. (F) Western blot assays to probe for IκBβ and tubulin in p65−/− MEFs transfected with vector control or catalytically inactive IKKα or IKKβ proteins (top). Inhibitory activity of IKK proteins was verified by luciferase NF-κB reporter assays (bottom). (G) p65−/− MEFs were transfected with either HA-tagged wild-type (WT) IκBβ or HA-tagged IκBβ mutated at serines 19 and 23 to either alanine (S19/23A) or glutamic acid (S19/23E) residues. After 24 h, cells were treated with cycloheximide (10 μg/ml) for the indicated times. Whole-cell extracts were then prepared and Western blot assays performed to probe for HA and α-tubulin.

Article Snippet: Briefly, p65 +/− TNF -α +/+ mice were crossed to p65 +/+ TNF -α −− mice (Jackson Laboratories, Bar Harbor, Maine).

Techniques: Real-time Polymerase Chain Reaction, Incubation, Labeling, Immunoprecipitation, Control, Polyacrylamide Gel Electrophoresis, Western Blot, Kinase Assay, Transfection, Plasmid Preparation, Activity Assay, Luciferase

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: IκBβ stability is regulated by the carboxyl terminus of p65. (A) p65−/− MEFs were infected with either vector control retrovirus vector or virus expressing wild-type p65. Whole-cell extracts were prepared from a mixed population of drug selection-resistant cells, and Western blot assays were performed to probe for p65 and IκBβ. (B) Western blot assays to probe for κB-Ras and β-arrestin in p65+/+ and p65−/− MEFs. α-Tubulin was used as a loading control. (C) Western blot assays were performed with p65+/+ TNF-α−/− and p65−/− TNF-α−/− tissue homogenates to probe for κB-Ras. (D) Illustration of full-length and C-terminal truncation mutants generated in p65. The mutants are named according to the fragment of p65 expressed. For example, p65(Δ534) denotes p65 containing amino acids 1 to 534. (E) Western blot assays for IκBβ (top) and p65 (bottom) in p65−/− MEFs transfected with the indicated p65 truncation mutants. (F) Western blot assay for IκBβ in p65(Δ313)-expressing brain, skeletal muscle (skmc), heart, and testis cells from a p65+/+ TNF-α−/− mouse. Arrows denote forms IκBβ-I and IκBβ-IV. (G) Identical lysates as described in panel E were either treated or not treated with phosphatase (PPase), and Western blot assays were subsequently performed to probe for IκBβ. (H) p65−/− MEFs were transfected with p65(Δ431) or p65(Δ313) and subsequently treated with MG-132 or not treated. Western blot assay was then performed to probe for IκBβ.

Article Snippet: Briefly, p65 +/− TNF -α +/+ mice were crossed to p65 +/+ TNF -α −− mice (Jackson Laboratories, Bar Harbor, Maine).

Techniques: Infection, Plasmid Preparation, Control, Virus, Expressing, Selection, Western Blot, Generated, Transfection

Journal:

Article Title: RelA/p65 Regulation of I?B?

doi: 10.1128/MCB.25.12.4956-4968.2005

Figure Lengend Snippet: MEFs stably expressing IκBβ exhibit a growth defect and increased apoptosis. p65−/− MEFs were infected with either a pBabe vector retrovirus or a virus expressing a degradation-resistant form of IκBβ tagged with an HA epitope (pBabeIκBβ-SR). (A) Extracts from p65−/−-expressing vector or HA-IκBβ-SR cells were prepared, and Western blot assays were performed to probe for HA and α-tubulin. (B) p65−/− cells expressing either the vector control or IκBβ-SR were treated with TNF-α, and at the indicated times nuclear extracts were prepared for EMSA. A supershift EMSA was performed with antibodies against p50, p65, or c-Rel to confirm the composition of NF-κB complexes (arrowheads denote supershifted subunits). (C) p65−/− MEFs were infected with a vector control or IκBβ-SR-expressing virus. Following 4 days of cell expansion under 1-μg puromycin selection, the cell number was determined in both cell lines. Cell number was normalized to vector control cells, which was set to a value of 100% growing cells. (D) Growth curves of p65+/+ or p65−/− cells expressing either a vector control or IκBβ-SR. (E) Growth curves identical to those performed in panel B from p65−/− cells infected with a vector control, IκBα-SR, or IκBβ-SR pBabe retrovirus. Cell number was normalized to the vector, which was set to a value of 100%. (F) Cells infected as in panel E were expanded under puromycin selection for 2 days and subsequently stained for Annexin V.

Article Snippet: Briefly, p65 +/− TNF -α +/+ mice were crossed to p65 +/+ TNF -α −− mice (Jackson Laboratories, Bar Harbor, Maine).

Techniques: Stable Transfection, Expressing, Infection, Plasmid Preparation, Virus, Western Blot, Control, Selection, Staining

Journal:

Article Title: Inhibition of NF-?B potentiates amyloid ?-mediated neuronal apoptosis

doi:

Figure Lengend Snippet: TNF-α is activating NF-κB in granule cells. (A) Concentration dependence. Granule cells were left untreated (0) or incubated for 24 h with TNF-α at the concentrations indicated. The TNF-α-mediated increase in nuclear NF-κB p65 immunoreactivity is depicted as means ± SEM (n > 10 neurons). A treatment with 2 ng/ml TNF-α induces a long-lasting robust increase in nuclear NF-κB immunoreactivity (P < 0.001 to other concentrations). (B) Pretreatment with TNF-α effects survival of granule cells. Granule cells were pretreated with TNF-α at the concentrations indicated, (200 units/ml, which is 2 ng/ml, or 400 units/ml, which is 4 ng/ml) followed by additional 24-h incubation in the presence of 10 μM Aβ. Note: only the NF-κB-activating dose (2 ng/ml or 200 units/ml) leads to neuroprotection (Left). Cell cultures were analyzed by indirect immunofluorescence for an increase of α-p65 mAb immunoreactivity (Upper), and nuclear DAPI staining is shown below. Scale bar, 25 μm. (C) Quantification of neuronal cell death after Aβ treatment. Data are shown as mean ± SEM of five independent determinations. Treatment scheme was as shown in Fig. ​Fig.11A. Treatment with 10 μM of Aβ-(1–40) after a pretreatment with 4 ng/ml TNF-α induced a > 40% reduction of viable cells (P < 0.001 to all other conditions), whereas pretreatment with 2 ng/ml TNF-α reverses the neurotoxic effect.

Article Snippet: Cells were fixed for 2 min in ethanol and for 5 min in 3.7% formaldehyde and immunostained with an α-p65 mAb (Boehringer-Mannheim) detected with a biotinylated α-mouse antibody decorated with streptavidin Cy3.

Techniques: Concentration Assay, Incubation, Immunofluorescence, Staining