william sellers  (Addgene inc)

Journal: The FEBS journal

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1111/febs.16375

Figure Lengend Snippet: AKT1 Phosphorylates HSF1 on S326 and induces HSF1 activity independent of mTORC1. (A-C) Indicated cell lines were transfected with vector, AKT1, AKT2 or AKT3 along with the HSE luciferase reporter for 48 h. Firefly/Renilla ratios were compared across groups. (D) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (E) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cell lysates were subjected to SDS/PAGE and immunoblotting with indicated antibodies. (F-G) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 in the presence or absence of rapamycin (10–20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunoblotting with indicated antibodies (G). Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

Article Snippet: The pcDNA3 Myr HA AKT3 plasmid was purchased from Addgene (ID 9017, RRID:Addgene_9017), which was originally established by Dr William Sellers.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Recombinant, Incubation, SDS Page, Western Blot, Reporter Assay

Journal: The FEBS journal

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1111/febs.16375

Figure Lengend Snippet: AKT1 promotes HSF1 trimerization and DNA binding. (A-B) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS/PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). (C) HEK-293 cells were transfected with vector, AKT1, AKT2 or AKT3 for 48 h. Cells were fixed with formaldehyde, lysed and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. (D-E) HEK-293 cells were transfected with either empty vector, AKT1, AKT2 or AKT3 for 48 h. Total RNA was collected from cells and subjected to RT-qPCR for the indicated genes. Experiments were completed in triplicate and analysed using one-way ANOVA and Tukey’s post-hoc test. Data are presented as mean ± SEM. * P < 0.05.

Article Snippet: The pcDNA3 Myr HA AKT3 plasmid was purchased from Addgene (ID 9017, RRID:Addgene_9017), which was originally established by Dr William Sellers.

Techniques: Binding Assay, Transfection, Plasmid Preparation, SDS Page, Western Blot, Sonication, Control, Quantitative RT-PCR

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: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

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: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

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: 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: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

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

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, but not Akt1or Akt2, significantly increased neurite formation in Neuro-2A cells. The relative efficiency of β-Gal+ cells containing neurites was calculated by dividing the number of β-Gal+ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal+ cells. The % of β-Gal+ cells with neurites in the control was set at 1. The other samples were compared to the control to obtain the relative efficiency of neurite formation. The average of three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing neurites following transfection with the Akt family member relative to the number of β-Gal+ Neuro-2A cells with neurites following transfection with an empty vector, as determined by the one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) multiple means comparison tests.

Article Snippet: A plasmid that expresses Akt3 was a gift from William Sellers (1236 pcDNA3 Myr HA Akt3, plasmid 9017; Addgene).

Techniques: Standard Deviation, Transfection, Plasmid Preparation