Journal: The Journal of Biological Chemistry

Article Title: Mineralocorticoid Receptor (MR) trans -Activation of Inflammatory AP-1 Signaling

doi: 10.1074/jbc.M116.732248

Figure Lengend Snippet: Expression of GR and MR in HEK293 cells. HEK293 cells were transfected with MR-DDK-myc or GR-DDK-myc expression vectors or empty vector. A, GR was expressed at low but detectable baseline levels, whereas MR was undetectable. Whole cell lysate Western blots demonstrate human GR and MR overexpression. Equal loading and transfer of protein were confirmed by α-tubulin expression. B, GR was overexpressed at higher levels compared with MR as shown by staining against a common DDK (FLAG) carboxyl-terminal tag. MR or GR cis-activation of an HRE-driven MMTV-luciferase reporter with dexamethasone (n = 3) (C), budesonide (n = 4) (D), deoxycorticosterone acetate (n = 3) (E), or aldosterone (n = 8; 0.01–1000 nm) (F). Luciferase activity was normalized to the activity of the Renilla control and presented as mean steroid ligand induction ±S.E. Luciferase activity of control plasmid-transfected cells is expanded as an inset. Error bars represent S.E.

Article Snippet: In addition, cells were simultaneously transfected with a luciferase reporter regulated by one of the following promoters: MMTV (pGL4.36(MMTV/luc2P/Hygro), Promega), NFκB (pGL 4.32(luc2P/NFκB-RE/Hygro, Promega), or AP-1, either sequence version 1 (AP-1_v1; 5′-TGAGTCAG; pGL4.44(luc2P/AP-1-RE/Hygro), Promega) or version 2 (AP-1_v2; 5′-TGACTAA; pGL4 custom/luc2P(AP-1-REv/Hygro), Promega), each with identical backbones but driven by six copies of different AP-1 consensus sequences.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Over Expression, Staining, Activation Assay, Luciferase, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Mineralocorticoid Receptor (MR) trans -Activation of Inflammatory AP-1 Signaling

doi: 10.1074/jbc.M116.732248

Figure Lengend Snippet: MR and GR regulation of AP-1 differs across various ligands. A, DOC (100 nm) activation of a PMA-induced (0.1–100 nm) AP-1 (AP-1_v1; (5′-TGAGTCAG)6) reporter in MR-transfected HEK293 cells. Maximal activation of the AP-1 reporter was seen at 10–100 nm PMA in the presence and absence (vehicle) of DOC. Luciferase activity was normalized to the activity of the Renilla control and presented as mean PMA induction (±S.E.; n = 4). Ligand dose-response curves (1–1000 nm) for DOC (n = 5) (B), aldosterone (n = 5) (C), dexamethasone (n = 5) (D), budesonide (n = 4) (E), or RU486 (n = 3) (F) of PMA-induced (100 nm) AP-1 reporter activity are shown. HEK293 cells were transfected with either MR, GR, or empty vector along with AP-1_v1 and Renilla reporters. Luciferase activity was normalized to the activity of the Renilla control and presented as mean PMA induction (set at 100% activity ±S.E.). G, MR or GR cis-activation of an HRE-driven MMTV-luciferase reporter is shown for various ligands (1 μm). Luciferase activity was normalized to the activity of the Renilla control and presented as mean steroid ligand induction (±S.E.; n = 3). Error bars represent S.E.

Article Snippet: In addition, cells were simultaneously transfected with a luciferase reporter regulated by one of the following promoters: MMTV (pGL4.36(MMTV/luc2P/Hygro), Promega), NFκB (pGL 4.32(luc2P/NFκB-RE/Hygro, Promega), or AP-1, either sequence version 1 (AP-1_v1; 5′-TGAGTCAG; pGL4.44(luc2P/AP-1-RE/Hygro), Promega) or version 2 (AP-1_v2; 5′-TGACTAA; pGL4 custom/luc2P(AP-1-REv/Hygro), Promega), each with identical backbones but driven by six copies of different AP-1 consensus sequences.

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Mineralocorticoid Receptor (MR) trans -Activation of Inflammatory AP-1 Signaling

doi: 10.1074/jbc.M116.732248

Figure Lengend Snippet: MR and GR DNA-binding domain mutations reverse MR activation of AP-1 and increase GR repression. A, alignment of human and rat MR and GR sequences. Amino acid differences are highlighted, and the lysine residue mutated in B–D is boxed. B, GR and GR-K442A or MR and MR-K624A with DEX or DOC (0.1–1000 nm), respectively, cis-activation of an HRE-driven MMTV-luciferase reporter. HEK293 cells were transfected with GR, the GR-K442A mutant, MR, the MR-K624A mutant, or empty vector as indicated along with MMTV and Renilla reporters. Luciferase activity was normalized to the activity of the Renilla control and presented as mean steroid ligand induction (±S.E.; n = 3). Luciferase activity of control plasmid- and mutant plasmid-transfected cells are expanded as insets. DEX and DOC dose-response curves (1–1000 nm) of PMA-induced (100 nm) reporter activity are shown for AP-1_v1 ((5′-TGAGTCAG)6) (C) or AP-1_v2 ((5′-TGAGTCAG)6) (D). Luciferase activity was normalized to the activity of the Renilla control and presented as mean PMA induction (set at 100% activity ±S.E.; n = 3). Error bars represent S.E.

Article Snippet: In addition, cells were simultaneously transfected with a luciferase reporter regulated by one of the following promoters: MMTV (pGL4.36(MMTV/luc2P/Hygro), Promega), NFκB (pGL 4.32(luc2P/NFκB-RE/Hygro, Promega), or AP-1, either sequence version 1 (AP-1_v1; 5′-TGAGTCAG; pGL4.44(luc2P/AP-1-RE/Hygro), Promega) or version 2 (AP-1_v2; 5′-TGACTAA; pGL4 custom/luc2P(AP-1-REv/Hygro), Promega), each with identical backbones but driven by six copies of different AP-1 consensus sequences.

Techniques: Binding Assay, Activation Assay, Luciferase, Transfection, Mutagenesis, Plasmid Preparation, Activity Assay