ser 15 phosphorylated p53  (Cell Signaling Technology Inc)

Journal: Cells, tissues, organs

Article Title: Engineered Aging Cardiac Tissue Chip Model for Studying Cardiovascular Disease.

doi: 10.1159/000516954

Figure Lengend Snippet: Fig. 2. Molecular characterization of the se- nescent phenotype. a Representative fluo- rescence images showing ROS activity in control and senescent cells. H9c2 cells were treated with 10 μM of CM-H2DCFDA and incubated for 60 min. Green fluorescence produced by the free radicals was captured by fluorescence microscopy. b Quantifica- tion of ROS fluorescence images showing ROS generation in control (Ctrl) and se- nescent (Sen) cells (n = 5,681 control nu- clei, n = 4,094 senescent nuclei, * p < 0.0001). c, d Quantification of total and phosphorylated p53 in control and senes- cent cells. H9c2 cells were fixed in formalin and immunostained using anti-p53 and anti-p53 (Ser15) antibodies, and the ex- pression level was assessed with a fluores- cence microscope (n = 11,914 control cells, n = 10,418 senescent cells, * p < 0.0001). e Fluorescence micrograph of control and senescent cells immunolabelled for the se- nescent marker p16INK4a (green) and F-ac- tin filaments (red).

Article Snippet: The cells that reached senescence and irreversible growth arrest were evaluated by the expression of the senescence-associated protein p16INK4a (Abcam, ab54210), transcription factor p53 (Cell Signaling Technology; 2527), and ser 15-phosphorylated p53 (Cell Signaling Technology, 9286).

Techniques: Activity Assay, Control, Incubation, Fluorescence, Produced, Microscopy, Marker

Journal: Frontiers in Molecular Neuroscience

Article Title: Loss of DEK Expression Induces Alzheimer’s Disease Phenotypes in Differentiated SH-SY5Y Cells

doi: 10.3389/fnmol.2020.594319

Figure Lengend Snippet: DEK knockdown in SH-SY5Y cells increases cell death via apoptosis. (A,B) DEK is successfully knocked down in SH-SY5Y cells using an shRNA lentiviral construct. A non-targeted shRNA (NTsh) was used as a control. (C) Number of dead cells were counted using Trypan blue. Neuronal differentiation exacerbated cell death due to DEK loss (** p < 0.01). (D) DEKsh cells showed increased phosphorylated p53 and cleaved caspase 3 (CC3) and 8, indicative of apoptosis. (E) Representative immunofluorescence images display increased CC3 in DEKsh cells. (F) Total β-catenin expression levels decreased in differentiated DEKsh cells. Activated β-catenin is downregulated in both undifferentiated and differentiated DEKsh cells.

Article Snippet: The membrane was blocked in 5% milk solution in TNET and incubated in primary antibodies including β-catenin (D10A8, 1:1,000, Cell Signaling), active β-catenin (non-phospho S45, 1:1,000, Cell Signaling) CC3 D175 (1:1,000; Cell Signaling), caspase 8 (1C12, 1:1,000; Cell Signaling), phosphorylated Ser 15 p53 (1:1,000; Cell Signaling), AT180 (1:500; Invitrogen) AT8 (1:500; Invitrogen), Phosphorylated Tau S262 (1:1,000; Abcam), PHF-1 (1:1,000; Invitrogen), DEK (1:1,000; ProteinTech rabbit polyclonal or 1:1,000 BD Biosciences, mouse monoclonal), Tau-1 (1:1,000; Sigma–Aldrich, mouse), Tau-5 (1:1,000; Abcam, mouse), and Actin C4 (1:10,000; mouse; gift of James Lessard, Cincinnati Children’s Hospital, available at Seven Hills Bioreagents).

Techniques: Knockdown, shRNA, Construct, Control, Immunofluorescence, Expressing

Journal: Aging (Albany NY)

Article Title: Nectandrin B-mediated activation of the AMPK pathway prevents cellular senescence in human diploid fibroblasts by reducing intracellular ROS levels

doi: 10.18632/aging.102013

Figure Lengend Snippet: NecB treatment reduced SA-β-gal activity and senescence marker expression in old HDFs. ( A ) Young (Y) and old (O) HDFs were treated with vehicle or 10−20 μg/mL NecB for 2 days and stained with X-gal to examine the activity of SA-β-gal. The stained cells were photographed under an inverted microscope (100× magnification). ( B ) The number of SA-β-gal-stained cells in A was counted and its percentage was plotted as the mean ± SEM. **P < 0.01, ***P < 0.001, compared with vehicle-treated control. ( C ) The protein levels in vehicle-treated (N0), 10 and 20 μg/mL NecB-treated (N10 and N20, respectively) young and old HDFs were compared by western blot analysis for senescence markers, including caveolin-1, P-Ser 15 -p53, p53, p21 waf1 , p16 ink4a , p27 kip1 , and cyclin D1. β-actin was used as an internal control. ( D ) The band density was examined by densitometry and normalized to β-actin. The relative protein levels were calculated and plotted as the mean ± SEM.

Article Snippet: Polyclonal antibodies against caveolin-1, phosphorylated P-Ser 15 -p53, p21 waf1 , and p16 ink4a , total AMPK, P-Thr 172 -AMPK, total mTOR, P-Thr 2446 -mTOR, raptor, total p70S6K, P-Thr 389 -p70S6K, total 4E-BP1, P-Thr 37/46 -4E-BP1, total p85, P-Tyr 458 -p85, total Akt, P-Ser 473 -Akt, total p38, P-Thr 85 -p38, total ASK1, P-Ser 83 -ASK1 (inactive form), and P-Thr 845 -ASK1 (active form) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Marker, Expressing, Staining, Inverted Microscopy, Control, Western Blot

Journal: Oncology Letters

Article Title: Liver kinase B1/adenosine monophosphate-activated protein kinase signaling axis induces p21/WAF1 expression in a p53-dependent manner

doi: 10.3892/ol.2018.8741

Figure Lengend Snippet: LKB1/AMPK requires p53 to regulate p21/WAF1. (A) P53 protein expression level in P53 wide-type HCT116 colon cell line and the isogenic HCT116 p53−/− cells. (B) P53 wild-type HCT116 and the isogenic HCT116 p53−/− cells were transiently transfected with control siRNA or LKB1 siRNA, and LKB1 and p21/WAF1 expression levels were analyzed using western blotting, with GAPDH used as a loading control. (C) HCT116 cells were transfected with plasmids encoding wild-type LKB1, LKB1 K78M or vector, and western blotting was performed to analyze p-p53 Ser15 and p21/WAF1 expression levels. GAPDH serves as the loading control. *P<0.05 vs. the control. Each blot is representative of three blots obtained from three independent experiments. Results are presented as the mean ± standard deviation. siRNA, short interfering RNA; LKB1, liver kinase B1; AMPK, adenosine monophosphate protein kinase; WAF1, cyclin dependent kinase inhibitor 1A.

Article Snippet: Antibodies against p21/WAF1, phosphorylated (p)-p53-Ser 15 and p-AMPK-Thr 172 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Transfection, Control, Western Blot, Plasmid Preparation, Standard Deviation, Small Interfering RNA