ikkβ flag  (Addgene inc)

Journal: bioRxiv

Article Title: The reovirus σ3 protein induces proteasomal degradation of IKKβ to modulate the host response to infection

doi: 10.64898/2026.01.08.698360

Figure Lengend Snippet: T3A σ3 associates with IKKβ. (A,B) HEK293T were transfected with 2 μg of IKKβ-Flag expression vector and either 3 μg of empty vector or vector expressing T3D L -σ3, T3A-σ3, or σNS. Following incubation at 37°C for 24 h, the cells were treated with 2 μM MLN4924 and incubated for another 20 h. The cells were harvested and subjected to immunoprecipitation with α-flag (A) or α-σ3 (B) antibody. The input and the IP lysates were immunoblotted with α-σ3, α-flag, σ-β-actin, or α-σNS antibody. (C) L929 were infected with T3D L /T3A S4 at MOI 10. After adsorption, the cells were treated with MLN4924 or DMSO at dose of 2μM. At 24h, cells were immunolabeled with σ3 (red), IKKβ (green) and host cell nuclei (blue) and finally imaged with a confocal microscopy. Images were deconvolved using Huygens Essential (17.10) software and scale bar (10 µm) were added using ImageJ software. Images are representative of three independent experiments.

Article Snippet: Expression vector for IKKβ-flag was purchased from Addgene (23298).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Infection, Adsorption, Immunolabeling, Confocal Microscopy, Software

Journal: bioRxiv

Article Title: The reovirus σ3 protein induces proteasomal degradation of IKKβ to modulate the host response to infection

doi: 10.64898/2026.01.08.698360

Figure Lengend Snippet: The presence of μ1 prevents IKKβ turnover by T3A-σ3. (A) HEK293T were transfected with 1.2 μg of empty vector or vector expressing T3DL σ3 or T3A σ3 along with 0.8 μg of expression vector for T3D F -μ1. 48 h after transfection, the cells were harvested and immunoblotted using α-σ3, α-flag, and α-β-actin antibodies (B) HEK293T were transfected with transfected with 3 μg of empty vector or vector expressing T3DL σ3 or T3A σ3 along with 2μg of IKK-β flag expression vector and 1 μg of T3D-μ1 expression vector. Following incubation at 37°C for 24 h, the cells were treated with 2 μM MLN4924 and incubated for an additional 20h. The cells were harvested and subjected to immunoprecipitation with α-flag antibody to precipitate IKKβ-flag. (A,B) The cells lysates were harvested and immunoblotted using α-σ3, α-flag, and α-β-actin, or α-reovirus antibodies.

Article Snippet: Expression vector for IKKβ-flag was purchased from Addgene (23298).

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Fbxo16 mediates degradation of NF-κB p65 subunit and inhibits inflammatory response in dendritic cells

doi: 10.3389/fimmu.2025.1524110

Figure Lengend Snippet: Fbxo16 forms a CRL1 complex, binds to p65 and negatively regulates NF-κB signaling. (A) Interaction of Fbxo16 and Cullins in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-Flag, and immunoblotted with anti-Cullin 1, Cullin 2 or Cullin 3. (B) Interaction of Fbxo16 and Skp1 in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Skp1. (C) Interaction of Fbxo16 and p65 in whole cell lysates of HEK293T cells, transfected with a Flag-p65 or p50 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-p65 or p50. (D) Interaction of Fbxo16 and PDLIM2 in whole cell lysates of HEK293T cells, transfected with a Flag-PDLIM2 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Flag. (E) Luciferase assay in MEFs transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs along with increasing amounts (wedge) of Fbxo16, then left untreated or stimulated with LPS for 5 hr. (F) Luciferase assay in HEK293T cells transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs without or with MyD88, IKKβ, TRAF6 or p65 along with increasing amounts (wedge) of Fbxo16. Data are representative of three (A, B) , four (C, D) or three ( E, F ; means ± SD, **P<0.01) independent experiments.

Article Snippet: Expression plasmids for Flag-tagged murine p50 (#20018), murine TRAF6 (#21624), murine MyD88 (#13093) and human IKKβ (#23298) were purchased from Addgene.

Techniques: Transfection, Immunoprecipitation, Luciferase, Construct