prl-null renilla construct  (Promega)

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: The pRL-Null renilla construct (#E2271) was purchased from Promega.

Techniques: Transfection, Immunoprecipitation, Luciferase, 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 mediates the polyubiquitination and degradation of p65 through its F-box domain. (A) Ubiquitination assay of p65 in HEK293T cells transfected with His-ubiquitin (His-Ub), p65 and increasing amounts (wedge) of Fbxo16. The purified ubiquitinated proteins were analyzed by immunoblot with anti-p65. (B) Effect of Fbxo16 on soluble and insoluble nuclear p65 in HEK293T cells transfected with Flag-p65 and increasing amounts (wedge) of c-Myc-Fbxo16, analyzed by immunoblot with ant-Flag antibody. (C) Effect of proteasome inhibitor on PDLIM2-mediated p65 degradation in soluble and insoluble nuclear extracts of HEK293T cells, transfected with Flag-p65 without or with c-Myc-Fbxo16, then left untreated or treated for 4 h with MG132 (10μM) and analyzed by immunoblot with anti-Flag antibody. (D) The structure of the wild-type Fbxo16 protein and Fbxo16 mutant lacking the F-box domain (ΔF). (E) Effect of the deletion of F-box domain on the interaction of Fbxo16 and Cullin 1 (top) or PDLIM2 (bottom) in HEK293T cells, transfected without or with c-Myc-wild-type or ΔF Fbxo16, immunoprecipitated with anti-c-Myc and immunoblotted with anti-Cullin 1 antibody (top), or transfected with Flag-PDLIM2 along with or without c-Myc-wild-type or ΔF Fbxo16, immunoprecipitated with anti-c-Myc and immunoblotted with anti-Flag antibody (bottom). (F) Ubiquitination assay of p65 in HEK293T cells cotransfected with His-ubiquitin, p65 along without or with wild-type or ΔF Fbxo16. The purified ubiquitinated proteins were analyzed by immunoblot with anti-p65. (G) Effect of the deletion of F-box domain on Fbxo16-mediated p65 degradation in soluble and insoluble nuclear extracts of HEK293T cells transfected with Flag-p65, along without or with wild-type or ΔF Fbxo16 and analyzed by immunoblot with anti-Flag antibody. (H) Luciferase assay in HEK293T cells transfected with ELAM−1 luciferase reporter, pRL-Null renilla constructs and Flag-p65 without or with wild-type or ΔF Fbxo16. Data are representative of three (A–C , E–G) or three ( H ; means ± SD, **P<0.01) independent experiments.

Article Snippet: The pRL-Null renilla construct (#E2271) was purchased from Promega.

Techniques: Ubiquitin Proteomics, Transfection, Purification, Western Blot, Mutagenesis, Immunoprecipitation, Luciferase, Construct

Journal:

Article Title: Cytokines inhibit norepinephrine transporter expression by decreasing Hand2

doi: 10.1016/j.mcn.2011.01.008

Figure Lengend Snippet: Phox2a does not regulate transcription of the NET gene. (A) M17 and SH5Y cells were untreated or treated with CNTF and ChIP analysis was performed with a Phox2a antibody or a negative IgG. PCR reactions were done with primers specific for TH, NET, and DBH. Positive signals from input DNA are shown from both M17 cells (M) and SH5Y cells (S). (B) ChIP analysis was performed with M17 cells that were treated with DMSO (Con) or UO126 (UO) for 90 minutes and PCR reactions were done with primers specific for NET and DBH. (C) M17 cells were subjected to ChIP analysis using antibodies against CREB or c-fos as a positive control. Positive signals from input DNA are shown. PCR reactions were performed with primers specific for TH, NET and DBH, and all three promoters were pulled down with CREB and c-fos antibodies. (D) Cells were treated with control medium (Control) or CNTF (CNTF) for 2 days prior to transfection with luciferase reporter constructs (NET-Luc+pRLNull) and a control vector or Phox2a (Phox2a + CNTF). Cells were maintained for an additional two days in vehicle or CNTF prior to analysis of luciferase. Data are a ratio of firefly luciferase/renilla luciferase. There was no significant difference between CNTF treated cells and CNTF+Phox2a. (E) Cells were treated with vehicle for 2 days prior to transfection with luciferase reporter constructs (TH-fLuc+pRLNull) and a control vector or Phox2a (Phox2a). Cells were maintained for an additional two days prior to analysis of luciferase. Data are a ratio of firefly luciferase/renilla luciferase. (F) Cells were treated with DMSO (Vehicle) or UO126 for 2 days prior to transfection with NET-Luc and pRL-Null. Cells were maintained in vehicle or UO126 for two more days prior to analysis of luciferase. Data is shown as a ratio of firefly luciferase/renilla luciferase. (G) Cells were treated with DMSO (all conditions), CNTF, or CNTF + UO126 for 2 days prior to transfection and then maintained in drug treatments for two days after transfection. Data are a ratio of firefly luciferase/renilla luciferase. Data are mean values ± SEM of triplicate values. Each experiment was repeated at least 3 times. *p<0.05, **p<0.01 vs control.

Article Snippet: The pRL-null renilla luciferase construct and the Dual-Luciferase Reporter Assay system were purchased from Promega (Madison, WI).

Techniques: Positive Control, Control, Transfection, Luciferase, Construct, Plasmid Preparation

Journal:

Article Title: Cytokines inhibit norepinephrine transporter expression by decreasing Hand2

doi: 10.1016/j.mcn.2011.01.008

Figure Lengend Snippet: Gata3 is suppressed by CNTF and rescues NET transcription. CNTF suppresses Gata3 mRNA in sympathetic neurons (A) and decreases Gata3 protein (B) in M17 cells. Gata3 levels were normalized to β-Actin levels and shown as a percent of control (mean values ± SEM, n=3). (C) A representative western blot showing Gata3 (top panel) and β-Actin (bottom) protein levels. (D) M17 cells were treated for 2 days with vehicle (Control) or CNTF. Cells were then transfected with NET-Luc+pRL-Null to monitor transcription, together with a control vector or Gata3 expression plasmid (Gata3 + CNTF) and maintained for two more days in CNTF or control medium. Data are a ratio of firefly luciferase/renilla luciferase. Data are mean values ± SEM of triplicate values. Each experiment was repeated at least 3 times. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The pRL-null renilla luciferase construct and the Dual-Luciferase Reporter Assay system were purchased from Promega (Madison, WI).

Techniques: Control, Western Blot, Transfection, Plasmid Preparation, Expressing, Luciferase

Journal:

Article Title: Cytokines inhibit norepinephrine transporter expression by decreasing Hand2

doi: 10.1016/j.mcn.2011.01.008

Figure Lengend Snippet: Hand2 is suppressed by CNTF and rescues NET transcription. CNTF suppresses Hand2 mRNA in sympathetic neurons (A) and decreases Hand2 protein (B) in M17 cells. Hand2 levels were quantified in the same cells analyzed for Gata3, and Hand2 protein was normalized to the β-Actin shown in Fig 5C. Data shown are percent of control (mean values ± SEM, n=3). (C) A representative western blot showing Hand2 (top panel). (D) M17 cells were treated for 2 days with vehicle (Control) or CNTF. Cells were then transfected with NET-Luc+pRL-Null to monitor transcription, together with a control vector or Hand2 expression plasmid (Hand2 + CNTF) and maintained for two more days in CNTF or control medium. Data are a ratio of firefly luciferase/renilla luciferase. Data are mean values ± SEM of triplicate values. Each experiment was repeated at least 3 times. *p<0.05, **p<0.01.

Article Snippet: The pRL-null renilla luciferase construct and the Dual-Luciferase Reporter Assay system were purchased from Promega (Madison, WI).

Techniques: Control, Western Blot, Transfection, Plasmid Preparation, Expressing, Luciferase