ikkβ nf κb inhibitor  (Tocris)

Journal: Drug Design, Development and Therapy

Article Title: Dexamethasone-Loaded Thermosensitive Hydrogel Suppresses Inflammation and Pain in Collagen-Induced Arthritis Rats

doi: 10.2147/DDDT.S256850

Figure Lengend Snippet: The Primary Antibodies Used in Western Blot Analysis

Article Snippet: Membranes were blotted with primary antibodies specific for NF-κB p65, inhibitor of NF-κB α (IκBα), IκB kinase (IKK) α/β, GAPDH, respectively (1:1000, ) at 4°C for 16 hours followed by incubation of the corresponding secondary antibodies (1:10,000; both from Cell Signaling Technology) for one hour at room temperature.

Techniques: Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: Crosstalk between the Akt/mTORC1 and NF-κB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs

doi: 10.1038/s41401-019-0272-2

Figure Lengend Snippet: mTORC1 interacts with and phosphorylates IKKα and IKKβ separately. a Superimposition of the binding poses of IKKα and IKKβ with mTOR. IKKα and IKKβ are shown in pink and gray, respectively. mTOR is shown in blue, with its PI3K kinase domain (residues 2182–2516) in cyan. ATP and Mg2+ ions are represented by sticks and spheres. b Key residues within the binding interfaces of the mTOR-IKKα and mTOR-IKKβ complexes. The Connolly surface of the binding interface was colored according to its electrostatic potential using Discovery Studio scripts. The key residues are shown as sticks. The C atoms are colored cyan, pink, and gray for mTOR, IKKα, and IKKβ, respectively. The H, N, and O atoms are colored white, blue, and red, respectively. The important hydrogen-bonding and electrostatic interactions are shown by dashed black and green lines, respectively. c IP with anti-mTOR and anti-Raptor antibodies and immunoblotting with an anti-IKKα antibody (left panel) and IP with an anti-IKKα antibody and immunoblotting with anti-mTOR and anti-Raptor antibodies (right panel) using PASMCs cultured under hypoxic conditions. d IP with an anti-mTOR antibody and immunoblotting with an anti-IKKα antibody using PASMCs treated with shMock (left panel) or shRaptor (right panel) and cultured under hypoxic conditions. e IP with anti-mTOR and anti-Raptor antibodies and immunoblotting with an anti-IKKβ antibody (left panel) and IP with an anti-IKKβ antibody and immunoblotting with anti-mTOR and anti-Raptor antibodies (right panel) using PASMCs cultured under hypoxic conditions. f In vitro kinase assay using active mTOR and IKKα or IKKβ. Mass spectrometry was performed to identify the phosphorylation sites (n = 5)

Article Snippet: A total of 1 μg of an inhibitor of NF-κB (IκB) kinase (IKK) α or IKKβ was mixed with active mTOR (0.4 μg/60 μL reaction; Cell Signaling Technology, Danvers, MA, USA) in 10 × kinase buffer containing 10 μM ATP and incubated at 30 °C for 30 min.

Techniques: Binding Assay, Western Blot, Cell Culture, In Vitro, Kinase Assay, Mass Spectrometry, Phospho-proteomics

Journal: Acta Pharmacologica Sinica

Article Title: Crosstalk between the Akt/mTORC1 and NF-κB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs

doi: 10.1038/s41401-019-0272-2

Figure Lengend Snippet: Hypoxia-induced activation of mTORC1 enhances the kinase activities of IKKα and IKKβ. Primary PASMCs were (a) cultured under hypoxic conditions or (b) cultured under hypoxic conditions and treated with rapamycin. Western blotting was performed with the indicated antibodies. (c) mTOR or (d) Raptor was knocked down in primary PASMCs cultured under hypoxic conditions. Western blotting was performed with the indicated antibodies. Primary PASMCs were (e) cultured under hypoxic conditions or (f) cultured under hypoxic conditions and depleted of IKKα/β. Western blotting was performed with the indicated antibodies. All phospho-protein levels were measured by densitometry and normalized to that of total protein (n = 5). HEK293T cells were transfected with the NF-κB luciferase plasmid, cultured under hypoxic conditions, and depleted of (g) mTOR or (h) Raptor. NF-κB luciferase activity was analyzed (n = 5). The data are expressed as the means ± standard errors of the means. */#P < 0.05, **/##P < 0.01, ***/###P < 0.001

Article Snippet: A total of 1 μg of an inhibitor of NF-κB (IκB) kinase (IKK) α or IKKβ was mixed with active mTOR (0.4 μg/60 μL reaction; Cell Signaling Technology, Danvers, MA, USA) in 10 × kinase buffer containing 10 μM ATP and incubated at 30 °C for 30 min.

Techniques: Activation Assay, Cell Culture, Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Bone morphogenetic protein-2 exhibits therapeutic benefits for osteonecrosis of the femoral head through induction of cartilage and bone cells

doi: 10.3892/etm.2018.5941

Figure Lengend Snippet: Effects of rBMP-2/Fc on synovial cells isolated from experimental mice. The expression of (A) BMP-2 and (B) BMP-2R in synovial cells from a mouse model of osteonecrosis of the femoral head were determined by ELISA. The expression of (C) IL-6 (D) IL-10, (E) VEGF, (F) M-CSF and (G) RANKL in synovial cells were analyzed by ELISA. (H) The half-life periods of rBMP-2/Fc and BMP-2 were determined by pharmacokinetic methods. (I) The affinity of rBMP-2/Fc for BMP-2R was measured. *P<0.05, **P<0.01. BMP-2, bone morphogenetic protein-2; rBMP, recombinant bone morphogenetic protein; Fc, Fc fragment; BMP-2R, bone morphogenetic protein-2 receptor; Il, interleukin; VEGF; vascular endothelial growth factor; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; SI-OTFD, steroid-induced osteonecrosis of the femoral head; DEX, dexamethasone.

Article Snippet: Prior to western blot analysis the membranes were blocked with 5% skimmed milk for 1 h at 37°C and subsequently incubated at 4°C overnight with rabbit anti-mouse primary antibodies sourced from Sigma-Aldrich (Merck KGaA) directed against: NF-κB (cat. no. SAB4501987), p65 (cat. no. SAB4502615), inhibitor of NF-κB kinase-β (IKK-β; cat. no. SAB1300467) and NF-κB inhibitor-α (IκBα; cat. no. SAB4501997).

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Experimental and Therapeutic Medicine

Article Title: Bone morphogenetic protein-2 exhibits therapeutic benefits for osteonecrosis of the femoral head through induction of cartilage and bone cells

doi: 10.3892/etm.2018.5941

Figure Lengend Snippet: Analysis of the signaling pathways mediated by rBMP-2/Fc in synovial cells. (A) Analysis of the association between BMP-2 and NF-κB expression in synovial cells as determined by luciferase activity. (B) The viability of synovial cells following incubation with rBMP-2/Fc (10 mg/ml), BMP-2 (2 mg/ml) or PBS for different time periods as determined by the refractive index. (C) The growth rate of synovial cells following treatment with PBS, DEX or rBMP-2/Fc for 24, 48 and 72 has determined by an MTT assay. (D) The effects of rBMP-2/Fc on the apoptosis of synovial cells as determined by flow cytometry. Western blot analysis was performed to determine the protein expression levels of (E) NF-κB, (F) p65, IKK-β and IκBα in synovial cells following treatment with BMP-2R or rBMP-2/Fc. The protein expression of (G) ALP, Runx2, osterix and osteocalcin, and (H) pSmad-1, pSmad-5 and pSmad-8 in Saos-2 cells was determined via western blot analysis.*P<0.05, **P<0.01. BMP-2, bone morphogenetic protein-2; rBMP, recombinant bone morphogenetic protein; Fc, Fc fragment; BMP-2R, bone morphogenetic protein-2 receptor; NF, nuclear factor; IKK-β, inhibitor of NF-κB kinase-β; IκBα, NF-κB inhibitor-α; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; p, phosphorylated; DEX, dexamethasone.

Article Snippet: Prior to western blot analysis the membranes were blocked with 5% skimmed milk for 1 h at 37°C and subsequently incubated at 4°C overnight with rabbit anti-mouse primary antibodies sourced from Sigma-Aldrich (Merck KGaA) directed against: NF-κB (cat. no. SAB4501987), p65 (cat. no. SAB4502615), inhibitor of NF-κB kinase-β (IKK-β; cat. no. SAB1300467) and NF-κB inhibitor-α (IκBα; cat. no. SAB4501997).

Techniques: Expressing, Luciferase, Activity Assay, Incubation, MTT Assay, Flow Cytometry, Western Blot, Recombinant

Journal: Experimental and Therapeutic Medicine

Article Title: Bone morphogenetic protein-2 exhibits therapeutic benefits for osteonecrosis of the femoral head through induction of cartilage and bone cells

doi: 10.3892/etm.2018.5941

Figure Lengend Snippet: In vivo effects of rBMP-2/Fc on steroid-induced osteonecrosis of the femoral head in a mouse model. (A) The clinical symptoms of mice with steroid-induced osteonecrosis of the femoral head were used to determine an arthritic score. (B) Pain-associated behaviors of experimental mice following treatment with PBS, DEX or rBMP-2/Fc were determined by PWMT. The mRNA expression levels of (C) BMP-2R, (D) RANKL, (E) VEGF and (F) M-CSF in experimental mice with steroid-induced osteonecrosis of the femoral head were determined by reverse transcription-quantitative polymerase chain reaction.**P<0.01. BMP-2, bone morphogenetic protein-2; rBMP, recombinant bone morphogenetic protein; Fc, Fc fragment; DEX, dexamethasone; PWMT, paw withdrawal mechanical threshold; BMP-2R, bone morphogenetic protein-2 receptor; VEGF; vascular endothelial growth factor; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand.

Article Snippet: Prior to western blot analysis the membranes were blocked with 5% skimmed milk for 1 h at 37°C and subsequently incubated at 4°C overnight with rabbit anti-mouse primary antibodies sourced from Sigma-Aldrich (Merck KGaA) directed against: NF-κB (cat. no. SAB4501987), p65 (cat. no. SAB4502615), inhibitor of NF-κB kinase-β (IKK-β; cat. no. SAB1300467) and NF-κB inhibitor-α (IκBα; cat. no. SAB4501997).

Techniques: In Vivo, Expressing, Real-time Polymerase Chain Reaction, Recombinant