wild type akt1  (Addgene inc)

Journal: PLoS ONE

Article Title: Akt-Signal Integration Is Involved in the Differentiation of Embryonal Carcinoma Cells

doi: 10.1371/journal.pone.0064877

Figure Lengend Snippet: ( A ) The profiles of Akt activity and its substrates match the change of pluripotency factors during RA-induced F9 cell differentiation process. F9 cells were seeded on petri dishes, induced with RA (1 µM) and harvested at 0, 1, 2, 4, 6, 8, 12, 24, 48 or 72 h. Cell lysates were subjected to immunoblotting with antibodies of anti-Oct4, anti-Nanog, anti-SATB1, anti-Klf4, anti-Akt1, anti-phospho-Akt (S473), anti-phospho-Akt (T308), anti-phospho-Akt substrate and anti-GAPDH. ( B ) The F9 stable cell lines were induced in the presence of RA as in (A) and harvested at 0, 2, 4, 8, 12, 24, 48 or 72 h, Immunoprecipitates with anti-Oct4 were subjected to immunoblotting with anti-Oct4 and anti-phospho-Oct4 (T228). ( C ) SATB1 and SATB1S47D are more efficient than SATB1S47A with respect to Nanog repression. The F9 stable cell lines were induced in the presence of RA as in (A) and harvested at 12, 24, 48 or 72 h. Cell lysates were subjected to immunoblotting with anti-Nanog, anti-Oct4, anti-Klf4, anti-SATB1, anti-phospho-SATB1 and anti-GAPDH. ( D ) A schematic representation of the dynamic change of Nanog expression in is shown. ( E ) The F9 stable cell lines were induced as in (C) and quantitative RT-PCR was performed to analyze the transcription level of Nanog . Results are from three independent experiments. ( F ) The F9 stable cell lines were induced with RA for 0, 12 or 24h and SATB1 occupancy on Nanog locus was documented using ChIP assay. ( G and H ) The F9 stable cell lines were induced as in (C) and quantitative RT-PCR was performed for Bcl2 and Nestin , two differentiation genes. ( I ) A working model for Akt-involved pluripotency/differentiation switch. See Discussion for details. The error bars in (E), (F), (G) and (H) represent mean ± SD from three independent experiments. Student’s t-test was performed between wild-type SATB1 and SATB1S47A groups (*p<0.05; **p<0.01).

Article Snippet: Plasmids of pUSE-Akt1 (wild-type, WT), pUSE-MyrAkt1 (activated, N-terminal myristoylation, Myr) and pUSE-Akt1 K179M (dominant negative, DN) were purchased from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Cell Differentiation, Western Blot, Stable Transfection, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

doi: 10.1101/2020.05.13.094573

Figure Lengend Snippet: High substrate count of AKT1. A log-log plot of the portion of the kinases with K substrates (P (K) ) and the substrate count (K) depicts the scale-free distribution of K, i . e ., a linear relationship between log 2 (P (K) ) and log 2 (K). The red * symbol and text illustrate the high Akt1 substrate count. The insert table lists the top 10 high-substrate-count kinases, with AKT1 in bolded text.

Article Snippet: Wild type AKT1 plasmid was purchased from Addgene (Addgene plasmid pcDNA3-myr-HA-AKT1 1036).

Techniques:

Journal: bioRxiv

Article Title: A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

doi: 10.1101/2020.05.13.094573

Figure Lengend Snippet: Expression of AKT1 proteins from transfected plasmid vectors. A: HA-tag Western blot analysis of control/untransfected HCT116 cells (C) and HCT116 cells transfected with either the wild type (WT) or the mutant AKT1 expression plasmids (Mutant Akt1). The cells were treated with the A*TPγS-loaded LCP nanoparticles and then lysed. The cell lysates were alkylated by PNBM and subjected to Western blot analysis with the anti-HA-tag antibody. The experiment was repeated more than 3 times. A representative result is shown. B: Transfection and western blot analyses workflow.

Article Snippet: Wild type AKT1 plasmid was purchased from Addgene (Addgene plasmid pcDNA3-myr-HA-AKT1 1036).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Mutagenesis

Journal: bioRxiv

Article Title: A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

doi: 10.1101/2020.05.13.094573

Figure Lengend Snippet: In vivo thiophosphate-tagging of kinase substrates and its detection strategy. A: Wild type kinase utilizes normal ATP to phosphorylate its substrates. In contrast, the mutated kinase, with enlarged ATP binding pocket, accepts bulky A*TPγS and was able to transfer the thiophosphate group onto the substrates. B: Workflow for thiophosphate-labelling of kinase substrates.

Article Snippet: Wild type AKT1 plasmid was purchased from Addgene (Addgene plasmid pcDNA3-myr-HA-AKT1 1036).

Techniques: In Vivo, Binding Assay

Journal: bioRxiv

Article Title: A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

doi: 10.1101/2020.05.13.094573

Figure Lengend Snippet: Analyses of in vivo AKT1 auto-thiophosphorylation and thiophosphorylation of AKT1 substrate IKKα. A and B : The transfected HCT116 cell lysates were subjected to anti-thiophosphate-ester antibody immunoprecipitation. This was followed by HA-tag ( A ) or IKKα ( B ) Western blot analyses of the control/untransfected HCT116 transfected cell lysate (C) as well as the immunoprecipitants of the lysates of HCT116 cells transfected with either wildtype (WT) or mutant AKT1 (Mutant Akt1) expression plasmids. The same cell lysates as in were used. Please note that the control/untransfected HCT116 cell lysate was directly used without immunoprecipitation. The experiments was repeated 3 times. A representative result is shown. C: Schematic illustration of the experimental workflow of immunoprecipitation of thiophosphated proteins followed by Western blot analyses.

Article Snippet: Wild type AKT1 plasmid was purchased from Addgene (Addgene plasmid pcDNA3-myr-HA-AKT1 1036).

Techniques: In Vivo, Transfection, Immunoprecipitation, Western Blot, Control, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

doi: 10.1101/2020.05.13.094573

Figure Lengend Snippet: Blue silver comassie stained SDS-PAGE gel. The wells for the molecular weight markers (MW), the immunoprecipitant of wild type AKT1 expression plasmid transfected cell lysate (Wild Type) and the immunoprecipitant of mutant AKT1 expression plasmid transfected cell lysate (Mutant) are marked. The antibody (IgG) heavy and light chains are also marked. The experiment was repeated 4 times. A representative result is shown.

Article Snippet: Wild type AKT1 plasmid was purchased from Addgene (Addgene plasmid pcDNA3-myr-HA-AKT1 1036).

Techniques: Staining, SDS Page, Molecular Weight, Expressing, Plasmid Preparation, Transfection, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: The natural agent rhein induces β‐catenin degradation and tumour growth arrest

doi: 10.1111/jcmm.13346

Figure Lengend Snippet: GSK‐3β is involved in rhein‐induced degradation of β‐catenin and inhibition of cell growth. ( A ) HepG2 and Hela cells were treated with or without LiCl (10 mM) and rhein (60 μM) for 48 hrs, followed by Western blot analysis of β‐catenin. ( B ) HepG2 and Hela cells were transfected with GSK‐3β siRNA. Twenty‐four hours after transfection, the cells were treated with or without rhein (60 μM) for 48 hrs, followed by Western blot analysis of β‐catenin and GSK‐3β. ( C ) HepG2 cells were treated with or without rhein (100 μM) for indicated periods, followed by Western blot analysis. ( D ) In vitro kinase assays of immunoprecipitated GSK3β. HepG2 cells were treated with or without 100 μM rhein for 24 hrs, followed by immunoprecipitation with anti‐GSK3β antibody or normal IgG. The immunoprecipitates were subjected to in vitro kinase assays in the presence of a peptide substrate. The relative kinase activity of GSK3β was plotted. ** P < 0.01; *** P < 0.001, compared to the kinase activity of GSK3β immunoprecipitates from rhein‐untreated cells, which was set as 1. ( E ) HepG2 cells were transduced with FLAG‐tagged phosphorylation‐deficient mutant of β‐catenin (S33A, S37A, T41A, S45A), followed by treatment with or without rhein (100 μM) for 24 hrs. The endogenous β‐catenin and FLAG‐tagged phosphorylation‐deficient mutant of β‐catenin were detected by Western blotting. ( F ) HepG2 cells were transduced with wild‐type Akt1 (WT‐Akt1) or constitutively active Akt1 (CA‐Akt1), followed by treatment with or without rhein (100 μM) for 24 hrs. The effects of Akt and rhein on β‐catenin expression, Akt and GSK3β phosphorylation were detected by Western blotting. ( G ) HepG2 cells were transduced with negative control siRNA (siCtrl) or GSK3β siRNA (siGSK3β). Twenty‐four hours later, the cells were treated with or without rhein (60 μM) for another 24 hrs, followed by CCK8 assays. The relative cell growth rate was plotted. In parallel, the efficiency of GSK3β knockdown was detected by Western blotting. *** P < 0.001; ** P < 0.01, compared with siControl. (H) Hela cells were transduced with siCtrl or siGSK3β. Twenty‐four hours later, the cells were treated with or without rhein (60 μM) for another 48 hrs, followed by CCK8 assays. The relative cell growth rate was plotted. In parallel, the efficiency of GSK3β knockdown was detected by Western blotting. ** P < 0.01.

Article Snippet: Plasmids for wild‐type Akt1 (WT‐AKT1) and constitutively active Akt1 (CA‐AKT1) were purchased from Addgene.

Techniques: Inhibition, Western Blot, Transfection, In Vitro, Immunoprecipitation, Activity Assay, Transduction, Phospho-proteomics, Mutagenesis, Expressing, Negative Control, Knockdown