akt1 kinase dead mutant  (Addgene inc)

Journal: Journal of Virology

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

doi: 10.1128/jvi.00901-20

Figure Lengend Snippet: Figure 2. Akt1 and Akt2 impair GR mediated activation of MMTV-LTR promoter. 693

Article Snippet: 154 Akt1 is a serine/threonine protein kinase, and the Akt1 kinase dead mutant (Addgene, 1014 155 pcDNA3 T7, that contains 3 point mutations; K179M, T308A and S473A) (56) was used to 156 test whether kinase activity was important for inhibiting GR-mediated transcription.

Techniques: Activation Assay

Journal: Journal of Virology

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

doi: 10.1128/JVI.00901-20

Figure Lengend Snippet: Akt family members influence GR- and DEX-mediated transactivation of the IEtu1 collapsed promoter. (A) Neuro-2A cells were transfected with the IEtu1 collapsed promoter construct containing the firefly luciferase reporter gene (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the IEtu1 collapsed promoter (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg). or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped fetal bovine serum (FBS) at approximately 24 h after transfection, and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: The Akt1 kinase dead mutant (1014 pcDNA3 T7; Addgene) includes 3 points mutations, K179M, T308A, and S473A.

Techniques: Transfection, Construct, Luciferase, Mutagenesis, Plasmid Preparation, Incubation

Journal: Journal of Virology

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

doi: 10.1128/JVI.00901-20

Figure Lengend Snippet: Akt1 and Akt2 impair GR-mediated activation of the MMTV-LTR promoter. (A) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that express GR (1.0 μg), Akt1, Akt2, or Akt3 (1.0 μg). (B) Neuro-2A cells were transfected with the MMTV-LTR promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS at approximately 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: The Akt1 kinase dead mutant (1014 pcDNA3 T7; Addgene) includes 3 points mutations, K179M, T308A, and S473A.

Techniques: Activation Assay, Transfection, Construct, Mutagenesis, Plasmid Preparation, Luciferase, Incubation

Journal: Journal of Virology

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

doi: 10.1128/JVI.00901-20

Figure Lengend Snippet: Akt1 significantly reduces GR- and KLF15-mediated transactivation of the HSV-1 ICP0 promoter. (A) Neuro-2A cells were transfected with the HSV-1 ICP0 promoter construct (0.5 μg), plasmids that express Akt1 or Akt1m (1.0 μg), and a plasmid that expresses GR (1.0 μg). (B) Neuro-2A cells were transfected with the ICP0 promoter construct (0.5 μg) and, where indicated, plasmids that expressed GR (1.0 μg), KLF15 (1.0 μg), Akt1 (1.0 μg, 2.0 μg, or 3.0 μg), or Akt1 kinase mutant construct (3.0 μg). All transfections contained a plasmid that expresses Renilla luciferase (0.05 μg) to normalize firefly luciferase values. To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Cells were incubated with 2% stripped FBS 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested and protein lysate subjected to a dual-luciferase assay. The results are the average of 3 independent experiments, and error bars denote the standard error. Student’s t test was used for statistical analysis. ns, not significant; **, P < 0.01; ***, P < 0.001. (C) Neuro-2A cells were transfected with a plasmid that expresses Akt1 (1.0, 2.0 or 3.0 μg of the expression vector as denoted), and a plasmid that expresses GR (1.0 μg). Cells were incubated with 2% stripped FBS 24 h after transfection and then certain cultures were treated with DEX (10 μM). At 48 h after transfection, cells were harvested, cell lysate prepared, and Western blot analysis performed to detect GR and the loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Fifty μg of protein was loaded in each lane. The results are the average of 3 independent experiments. kd, kilodalton.

Article Snippet: The Akt1 kinase dead mutant (1014 pcDNA3 T7; Addgene) includes 3 points mutations, K179M, T308A, and S473A.

Techniques: Transfection, Construct, Plasmid Preparation, Mutagenesis, Luciferase, Incubation, Expressing, Western Blot, Control

Journal: Journal of Virology

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

doi: 10.1128/JVI.00901-20

Figure Lengend Snippet: Akt3 efficiently promotes neurite formation in Neuro-2A cells. Neuro-2A cells were cotransfected with an empty vector (pcDNA3.1) (A), a plasmid expressing Akt1 or Akt2 (Panel B), or Akt3 (C and D) (1 μg plasmid DNA) and a plasmid expressing the lacZ gene (0.1 μg plasmid) to mark transfected cells. (B) A typical result from cells transfected with Akt1 or Akt2. To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000 cells/cm2) and then incubated with minimal essential medium (MEM) that contained 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were detected by staining.

Article Snippet: The Akt1 kinase dead mutant (1014 pcDNA3 T7; Addgene) includes 3 points mutations, K179M, T308A, and S473A.

Techniques: Plasmid Preparation, Expressing, Transfection, Incubation, Staining