Journal: Journal of Biological Chemistry

Article Title: Protein Phosphatase 2Cβ Association with the IκB Kinase Complex Is Involved in Regulating NF-κB Activity

doi: 10.1074/jbc.m306273200

Figure Lengend Snippet: FIG. 1. PP2C associates with IKK complex. A, Western blot analysis with the FLAG antibody was performed on extracts prepared from G418-resistant 293 cells either in the absence (lane 1) or presence of FLAG-PP2C (lane 2). B, extracts from the 293 cells stably express- ing FLAG-tagged PP2C were immunoprecipitated (IP) with either normal mouse IgG (lane 1) or the FLAG antibody (lane 2) followed by Western blot analysis with mouse monoclonal antibodies directed against IKK (top) and IKK (middle) or a rabbit polyclonal antibody for IKK/NEMO (bottom). The extract input is also shown (lane 3). C, a CMV expression vector alone (lane 1) or encoding Myc-tagged wild type PP2C (2.0 g) was cotransfected into 293 cells with 2.0 g of either FLAG-tagged IKK (lane 2), IKK (lane 3), or IKK/NEMO (lane 4). Extracts were immunoprecipitated with either the FLAG antibody (top) or normal mouse IgG (middle), and Western blot analysis was per- formed with a Myc antibody to detect Myc-PP2C (lanes 1–5, top and middle). Western blot analysis of these extracts with the FLAG and Myc antibodies was also performed (lanes 1–4, bottom). D, extracts from HeLa cells were immunoprecipitated with either normal mouse IgG (lane 1) or a murine polyclonal antibody directed against PP2C (lane 2) followed by Western blot analysis with antibodies directed against IKK (top), IKK (middle), and IKK/NEMO (bottom). The extract input is also shown (lane 3). The bottom shows Western blot analysis of extracts prepared from HeLa cells either in the absence (lane 1) or presence of a transfected FLAG-PP2C construct (lane 2) using the murine polyclonal antibody directed against PP2C.

Article Snippet: Antibodies used in these studies included monoclonal antibodies directed against the M2 FLAG epitope (F-3165; Sigma), the Myc epitope (sc-40; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), IKK (556532; Pharmingen), and IKK (550621; Pharmingen) in addition to normal mouse IgG (sc-2025; Santa Cruz Biotechnology) and polyclonal antibodies directed against I B (sc-371; Santa Cruz Biotechnology), phosphoI B (9241; Cell Signaling), IKK (sc-7607; Santa Cruz Biotechnology), IKK /NEMO (sc-8330; Santa Cruz Biotechnology), IKK (sc-7218; Santa Cruz Biotechnology), influenza hemagglutinin (HA) epitope (sc805; Santa Cruz Biotechnology), or actin (A2066; Sigma).

Techniques: Western Blot, Stable Transfection, Immunoprecipitation, Bioprocessing, Expressing, Plasmid Preparation, Transfection, Construct

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