prl-null internal renilla luciferase control construct  (Promega)

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