ap-1-luc reporter plasmid  (Promega)

Journal: Cell reports

Article Title: Bioinformatics Screening of Genes Specific for Well-Regenerating Vertebrates Reveals c-answer , a Regulator of Brain Development and Regeneration

doi: 10.1016/j.celrep.2019.09.038

Figure Lengend Snippet: (A) Schematic of the experiment performed to analyze the effects of c-Answer on the expression of the MAP/ERK pathway luciferase reporter pSPE-Luc pGL4.33. The stress-activated reporter AP-1-Luc pGL4 was used as a control. (B) Diagram showing the Luc signal analysis results for the two reporters in the animal caps of embryos expressing the indicated proteins. (C) Schematic of the experiment performed to analyze the effects of c-Answer on the Ca 2+ flux in response to the addition of the P2ry1 agonist ADP to animal cap cells expressing the Ca 2+ sensor Case12 and purinergic receptor P2ry1. (D) Fluorescent images of cells expressing the indicated proteins before and after ADP addition. (E) Diagram showing the results of Case12 signal analysis in animal cap cells expressing the indicated proteins. Scale bars: 200 μm in (D).

Article Snippet: Embryos were injected at 2–4 cell stage with synthetic c-answer + fgf8 mRNAs or solely fgf8 mRNA (3–4 nL of 100ng/μl mRNA water solution per embryo) mixed with one of the luciferase reporter plasmids: AP-1-Luc pGL4.44[luc2P/AP1 RE/Hygro] (Promega) sensitive to the stress-activated MAPK/JNK pathway, or SRE-Luc pGL4.33[luc2P/SRE/Hygro] (Promega) sensitive to MAP/ERK pathway, and the reference pRenilla plasmid (50 pg/embryo of each reporter plasmid).

Techniques: Expressing, Luciferase

Journal: Cell reports

Article Title: Bioinformatics Screening of Genes Specific for Well-Regenerating Vertebrates Reveals c-answer , a Regulator of Brain Development and Regeneration

doi: 10.1016/j.celrep.2019.09.038

Figure Lengend Snippet:

Article Snippet: Embryos were injected at 2–4 cell stage with synthetic c-answer + fgf8 mRNAs or solely fgf8 mRNA (3–4 nL of 100ng/μl mRNA water solution per embryo) mixed with one of the luciferase reporter plasmids: AP-1-Luc pGL4.44[luc2P/AP1 RE/Hygro] (Promega) sensitive to the stress-activated MAPK/JNK pathway, or SRE-Luc pGL4.33[luc2P/SRE/Hygro] (Promega) sensitive to MAP/ERK pathway, and the reference pRenilla plasmid (50 pg/embryo of each reporter plasmid).

Techniques: Recombinant, CRISPR, Reporter Assay, Clone Assay, Plasmid Preparation, Software

Journal: Acta Pharmacologica Sinica

Article Title: BX-795 inhibits HSV-1 and HSV-2 replication by blocking the JNK/p38 pathways without interfering with PDK1 activity in host cells

doi: 10.1038/aps.2016.160

Figure Lengend Snippet: BX-795 inhibition of HSV replication by blocking JNK/p38 pathway. (A) HSV-2 infection stimulated JNK and p38 MAP kinase pathway, but not ERK pathway. (B) HSV-2 infection also activated the phosphorylation of c-Jun and ATF-2. Cells infected with HSV-2 (moi=1) were collected at the indicated time points. ERK, JNK, p38 MAP kinase, c-Jun and their phosphorylated forms and p-ATF-2 were determined by Western blot. (C) JNK and p38 MAP kinase inhibitors blocked HSV-2 replication. Confluent HEC-1-A cells or Vero cells were infected with HSV-2 (moi=1) in the presence of serial concentrations of SB203580, SP600125 or PD98059. gD expression was determined 24 h postinfection. (D) BX-795 inhibited HSV-2-induced activation of JNK and p38 MAP kinase pathways. (E) BX-795 inhibited further activation of c-Jun and ATF-2 caused by HSV-2 infection. HEC-1-A cells were either mock-infected or infected with HSV-2 (moi=1) in the presence or absence of BX-795. JNK, p38 MAP kinase, c-Jun and their phosphorylated forms and p-ATF-2 were determined 12 h postinfection. (F) BX-795 blocked PMA-stimulated c-Jun activation. HEC-1-A cells were either mock-treated or treated with PMA (5 μg/mL) in the presence or absence of BX-795. The cells were collected after 2 h treatment, and c-Jun and its phosphorylated form were determined. (G) BX-795 inhibited HSV-2-mediated c-Jun nuclear translocation. HEC-1-A cells were either mocked-treated or treated with PMA (5 μg/mL) or infected with HSV-2 (moi=1) in the presence or absence of BX-795. c-Jun nuclear translocation was determined by immunofluorescence staining 2 h post-treatment with PMA or 12 h post-infected with HSV-2. (H) BX-795 inhibited HSV-2-induced AP-1 activation in a dose-dependent manner. HEC-1-A cells were transfected with AP-1 luc reporter plasmid. After 24 h, the cells were either mock-infected or infected with HSV-2 (moi=1) in the presence of serial concentrations of BX-795. AP-1 activity was determined by luciferase assay.

Article Snippet: NF-κB-luc and AP-1-luc reporter plasmids were from Clontech (California, USA). pcDNA3-ICP0-1-GFP and pcDNA3-ICP0-2-GFP were kindly provided by Dr Claus-Henning Nagel of Heinrich Pette Institute – Leibniz Institute for Experimental Virology. pCMV3-p38-C-GFPSpark and pCMV3-JNK1-N-GFPSpark were obtained from Sino Biological Inc (Beijing, China).

Techniques: Inhibition, Blocking Assay, Infection, Western Blot, Expressing, Activation Assay, Translocation Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Activity Assay, Luciferase

Journal: Acta Pharmacologica Sinica

Article Title: BX-795 inhibits HSV-1 and HSV-2 replication by blocking the JNK/p38 pathways without interfering with PDK1 activity in host cells

doi: 10.1038/aps.2016.160

Figure Lengend Snippet: BX-795 inhibition of HSV replication by blocking JNK/p38 pathway. (I) BX-795 also inhibited PMA and TNF-α stimulated AP-1 activation. HEC-1-A cells were transfected with AP-1 luc reporter plasmid. After 24 h, cells were mock-treated, treated with PMA (5 μg/mL) or rhTNF-α (100 ng/mL) or infected with HSV-2 (moi=1) in the presence of BX-795 (25 μmol/L). AP-1 activity was determined. (J) BX-795 inhibition on HSV replication attenuated by overexpression of p38/JNK. HeLa cells with or without JNK1 or p38 overexpression were infected with HSV-2 and total cellular protein was prepared for WB analysis. gD-2, the phosphorylation level of p38 MAP kinase, JNK and their substrates were determined via Western blot.

Article Snippet: NF-κB-luc and AP-1-luc reporter plasmids were from Clontech (California, USA). pcDNA3-ICP0-1-GFP and pcDNA3-ICP0-2-GFP were kindly provided by Dr Claus-Henning Nagel of Heinrich Pette Institute – Leibniz Institute for Experimental Virology. pCMV3-p38-C-GFPSpark and pCMV3-JNK1-N-GFPSpark were obtained from Sino Biological Inc (Beijing, China).

Techniques: Inhibition, Blocking Assay, Activation Assay, Transfection, Plasmid Preparation, Infection, Activity Assay, Over Expression, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: BX-795 inhibits HSV-1 and HSV-2 replication by blocking the JNK/p38 pathways without interfering with PDK1 activity in host cells

doi: 10.1038/aps.2016.160

Figure Lengend Snippet: BX-795 inhibited ICP0-mediated AP-1 activation, but not NF-κB activation. (A) BX-795 inhibited ICP0-mediated AP-1 activation. HEC-1-A, Vero and 293T cells were co-transfected with pcDNA3-ICP0-1/2-GFP or pcDNA3 and AP-1 luc reporter plasmid. After 24 h, cells were either mock-treated or treated with BX-795 (25 μmol/L). The luciferase activities were measured 24 h post-treatment. (B) BX-795 did not impede ICP0-mediated NF-κB activation. HEC-1-A cells were co-transfected with pcDNA3-ICP0-1/2-GFP or pcDNA3 and NF-κB luc reporter plasmid. After 24 h culture, cells were either mock-treated or treated with BX-795 (25 μmol/L), BAY11-7082 (20 μmol/L), PDTC (10 μg/mL), PGA1 (10 μg/mL) or MG132 (20 μmol/L). The luciferase activities were measured 24 h post-treatment.

Article Snippet: NF-κB-luc and AP-1-luc reporter plasmids were from Clontech (California, USA). pcDNA3-ICP0-1-GFP and pcDNA3-ICP0-2-GFP were kindly provided by Dr Claus-Henning Nagel of Heinrich Pette Institute – Leibniz Institute for Experimental Virology. pCMV3-p38-C-GFPSpark and pCMV3-JNK1-N-GFPSpark were obtained from Sino Biological Inc (Beijing, China).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase