rabbit anti cip2a  (Novus Biologicals)

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Identification of transcription factor binding sites with matrix similarity of 95% and core similarity of 100% on −1802 bp CIP2A promoter using Genomatix software. B. Phylogenetic tree depicting the evolutionary conservation of CIP2A promoter. C. Identification of putative CpG Island from −150 bp to +400 bp (blue shaded area) on the CIP2A promoter using MethPrimer software. D. Shows the sequencing results of the extracted genomic DNA from normal human blood. All CpG sites (represented by black rectangular blocks) lying within the CpG island were converted from CG to TG when treated with bisulphite, thereby implying that CIP2A promoter at these sites is unmethylated.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Binding Assay, Software, Sequencing

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Identified single nucleotide polymorphisms on −1802 to +182 region of CIP2A promoter from indicated cells. Two cancer cell line specific changes were observed, namely G/A at −1101 in SCC-7 cell line and T/C at −592 in HeLa cell line. B. Relative Luciferase assay showing the relative activity of −1802 bp of CIP2A promoter in comparison to well known oncogenic promoters like EGFRLuc and 5xJunLuc. C. Luciferase assay showing comparison of activity between wild type (WT) CIP2A promoter and the indicated SNP mutants. D. Luciferase assay showing the activity of indicated CIP2A promoters. High basal activity is mediated by first 392 bp of the promoter, out of which, almost two-thirds of it was due to the first 108 bp. B–D, Shown is the Mean+SD from three independent experiments.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Quantitative PCR (qPCR) analysis showing levels of CIP2A mRNA extracted from AGS cells treated with DMSO, SB23580 (20 uM), LY294002 (10 uM), PD98059 (20 uM), AG1478 (10 uM) and TPA (100 nM) at 24 h time point. While a decrease in CIP2A mRNA level was observed with PD98059 and AG1478 treatments, TPA treatment augmented the CIP2A mRNA levels in AGS cells. B. qPCR analysis showing time-dependent regulation of CIP2A mRNA levels in AGS cells treated with DMSO, AG1478 (10 uM) and TPA (100 nM) for indicated time points. C and D Luciferase assays showing the activity of indicated CIP2A promoter deletions in cells treated either with DMSO, AG1478 (10 uM) or TPA (100 nM) for 24 h. (*, P <0.05; n.s. = non-significant). B–D. Shown is the Mean+SD from three independent experiments.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Western blot showing concentration dependent effect of specific MEK inhibitor, U0126, on CIP2A protein levels in AGS cells at 48 h time point. B. Western blot showing the effect of both MEK1 and MEK2 siRNAs on CIP2A protein levels in AGS cells at 72 h time point.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Luciferase assay showing the activity of different length CIP2A promoters when treated with DMSO and U0126 (10 uM) for 24 h, identifying the MEK responsive region to lie between −672 bp to −27 bp of the CIP2A promoter. B. Luciferase assay showing the basal activity indicated CIP2A promoters. C. Luciferase assays showing the activity of different length CIP2A promoters when treated with DMSO and U0126 (10 uM) for 24 h, further narrowing down the MEK responsive region to be between −108 bp and −27 bp of the CIP2A promoter (*, P <0.05; **, P <0.00; n.s. = non-significant). Shown is the Mean+SD from three independent experiments.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Schematic diagram of the 108 bp fragment of CIP2A promoter showing the location of two overlapping predicted ETS1 binding sites. B. Sequencing results of ETS-1 binding site mutants on the CIP2A promoter created using mutagenesis. The sequence on the top represents the wild type sequence (read from 5′ to 3′ end), while the sequence below is the mutated sequence (read from 5′ to 3′ end). The induced change in the sequence is shown within the rectangular box. C. Luciferase assay comparing the activity of either wild type −108CIP2ALuc or indicated ETS binding site mutant −108CIP2ALuc constructs. D,E&F. Luciferase assays comparing the activity of either wild type −108CIP2ALuc or ETS1 Site1 mutant −108CIP2ALuc constructs after treatment with DMSO, AG1478 (10 uM; D), UO126 (10 uM; E) or TPA (100 nM; F) for 24 h. (*, P <0.05; **, P <0.01; n.s. = non-significant). Shown is the Mean+SD from three independent experiments.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Construct

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A. Chromatin immunoprecipitation assay of ETS1 binding to CIP2A promoter in either DMSO or UO126 treated cells. UNQ9419 promoter is shown as a positive control. (*, P <0.05; **, P <0.01; n.s. = non-significant). B. Luciferase assay comparing the activity of either wild type −1802CIP2ALuc or indicated ETS binding site mutant −1802CIP2ALuc constructs. C,D&E. Western blot analysis of CIP2A and ETS1 protein levels in either AGS (C), PC-3 (D) or LNCaP (E) cells transfected with scrambled (Scr.) or ETS-1 specific siRNAs for 72 h.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Chromatin Immunoprecipitation, Binding Assay, Positive Control, Luciferase, Activity Assay, Mutagenesis, Construct, Western Blot, Transfection

Journal: PLoS ONE

Article Title: ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

doi: 10.1371/journal.pone.0017979

Figure Lengend Snippet: A,B,C&D. Oncomine database analysis of gene expression profiles for EGFR-MEK-ETS1 pathway genes revealed M6 subtype of acute myeloid leukemia as a cancer type in which CIP2A and representative genes of each level of the pathway are significantly upregulated (**, P <0.01).

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS-0.1%-NP40 and then incubated with mouse monoclonal MEK1 (H8), the mouse monoclonal MEK2 (A-1), the rabbit polyclonal ETS1 (C20), goat polyclonal anti-β-Actin (all from Santa Cruz, Biotechnology, Santa Cruz, CA, USA) or with rabbit polyclonal anti-CIP2A .

Techniques: Expressing

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 1. CIP2A is expressed in astrocytes and upregulated in AD brains. (A) Primary neurons and astrocytes from the rat hippocampus were cocultured for 2 days (DIV 2) and immunostained with antibodies against CIP2A (green), Map-2 (red), and Hoechst (blue). Scale bar ¼ 20 mm. (B) Rat primary astrocytes (DIV 3, 7, 14, and 21) were immunostained with antibodies against CIP2A (green), GFAP (red), and Hoechst (blue). Scale bar ¼ 40 mm. (C) Left: CIP2A and GFAP expression levels in the temporal cortex from patients with AD were detected by Western blotting. GAPDH was used as a loading control. Right: Quantitative analysis of the CIP2A and GFAP levels. *p < 0.05 versus control, n ¼ 5 control group, n ¼ 4 AD group. (D) Left: CIP2A and GFAP expression levels in the hippocampus from 3xTg-AD and control mice (male, 9 months old) were detected by Western blotting. GAPDH was used as a loading control. Right: Quantitative analysis of the CIP2A and GFAP levels. **p < 0.01, ***p < 0.001 versus WT mice, n ¼ 3. Data are mean SEM. Abbreviations: AD, Alzheimer’s disease; CIP2A, cancerous inhibitor of PP2A; GFAP, glial fibrillary acid protein. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 2. Overexpression of CIP2A induces reactive astrogliosis. Primary rat astrocytes (DIV15) were infected with AAV-CIP2A for 5 days. (A) Immunofluorescence staining of GFAP (red) and Hoechst (blue). Scale bar ¼ 40 mm. (B) The numbers of astrocytes were counted in control and CIP2A overexpression group. n ¼ 9 well per group. (C) Left: CIP2A and GFAP expression levels were detected by Western blotting. DM1A was used as a loading control. Right: Quantitative analysis of the CIP2A and GFAP levels. n ¼ 3 per group. (D) IL-1a, IL-6, and TNF-a in culture media were detected by ELISA, n ¼ 9 well per group. (E) Ab40 and Ab42 levels in culture media were detected by ELISA, n ¼ 9 well per group. *p < 0.05, **p < 0.01, ***p < 0.001 versus vector. Data are mean SEM. Abbreviations: CIP2A, cancerous inhibitor of PP2A; GFAP, glial fibrillary acid protein. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Infection, Staining, Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 3. CIP2A-induced astrogliosis promotes synaptic degeneration in primary neurons. Rat primary cortical neurons (DIV 10) were cultured in the conditional media from CIP2A- overexpressed astrocytes (DIV 10, infected with AAV-CIP2A for 5 days) for 4 days. (A) Cell viability was detected by LDH cytotoxicity assay kit. n ¼ 9 well per group. (B) Left: PSD95, synapsin I, and synaptophysin expression levels were detected by Western blotting using the antibodies indicated. DM1A was used as a loading control. Right: Quantitative analysis of the PSD95, synapsin I, and synaptophysin levels. *** p < 0.001 versus vector, n ¼ 3 per group. Data are mean SEM.

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Infection, LDH Cytotoxicity Assay, Expressing, Western Blot, Control, Plasmid Preparation

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 4. Overexpression of CIP2A in astrocytes induces visual episodic memory deficit in mice. (A) Timeline of the experiment. (B) The structure diagram of AAV-CIP2A. (C) AAV- CIP2A was injected into the hippocampal CA3 region of the C57/BL6 mice, and CIP2A expression was observed (green) 4 weeks after injection. Cell nuclei were stained with Hoechst (blue) to show the hippocampus. Scale bar ¼ 500 mm. (D) Brain slices in C were immunostained with anti-GFAP to show astrocytes; virus infection efficiency was obtained through calculating the ratio of costained (red and green) cells/GFAP-positive (red) cells. (E) The experimental design of novel object recognition test (NOR). One day before the memory acquisition, the mice were habituated to the arenas for 5 minutes, as indicated in the first box. The second box showed the acquisition trial. The third and forth box showed the test trial conducted 1 hour and 24 hours after the acquisition trial. (F) The recognition index of object A and B in the acquisition trial. (G) The recognition index of object A and C in the test trial 1 hour post the acquisition trial. *** p < 0.001 unfamiliar object C versus familiar object A. (H) The recognition index of object A and D in the test trial 24 hours post the acquisition trial. *** p < 0.001 unfamiliar object D versus familiar object A. (I) The discrimination index of 1 hour test trial and 24 hours test trial. *** p < 0.001 versus vector group. n ¼ 6 mice per group. Data are mean SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Injection, Expressing, Staining, Virus, Infection, Plasmid Preparation

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 5. Overexpression of CIP2A in astrocytes induces spatial memory deficit in mice. (A) The experimental design of Morris water maze test (MWM). (B) The representative searching trace on day 6 of the training. (C) The latency to reach the hidden platform. (D) Left: Swimming speed after removing the platform (1 hour) during the probe trial. Middle: The number of times of crossing the position of the platform. Right: The percentage of time spent in target quadrant. Quadrant 3 is the target quadrant. (E) Left: Swimming speed after removing the platform (48 hours) during the probe trial. Middle: The number of times to cross the position of the platform. Right: The percentage of time spent in target quadrant. *** p < 0.001 versus vector. n ¼ 6 mice per group. Data are mean SEM.

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Plasmid Preparation

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 6. Overexpression of CIP2A in astrocytes induces neuronal loss, synaptic degeneration, and impairment in hippocampal LTP in mouse brains. (A) Left: Nissl staining of the brain slices from Con, AAV-vector, and AAV-CIP2A infected mouse brains. Right: Quantitative analysis of the neuron numbers in CA1 and CA3 regions of the hippocampus. n ¼ 3 brain slices per mouse, 3 mice per group. (B) Top: Representative dendritic spines of neurons in hippocampal CA3 region from Golgi impregnated hippocampus. Bottom: Quantitative analysis of averaged spine density (mean spine number per 10 mm dendrite segment). n ¼ 10, from 3 mice per group. (C) Top: CIP2A, PSD95, synapsin I, and synaptophysin expression levels were detected by Western blotting in mouse cortex. DM1A was used as a loading control. Bottom: Quantitative analysis of the protein levels, n ¼ 3 per group. (D) Top: CIP2A, PSD95, synapsin I, and synaptophysin expression levels were detected by Western blotting in mouse hippocampus. DM1A was used as a loading control. Bottom: Quantitative analysis of the protein levels, n ¼ 3 per group. (E) Top: Normalized CA3-CA1 fEPSP mean slope recorded from the CA1 dendritic region in acute hippocampal slices. Bottom: Quantitative analysis of the normalized fEPSP slope. n ¼ 15, 3 mice per group, 5 brain slices per mouse were recorded. * p < 0.05, ** p < 0.01, *** p < 0.001 versus vector. Data are mean SEM.

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Staining, Plasmid Preparation, Infection, Expressing, Western Blot, Control

Journal: Neurobiology of aging

Article Title: CIP2A-promoted astrogliosis induces AD-like synaptic degeneration and cognitive deficits.

doi: 10.1016/j.neurobiolaging.2018.11.023

Figure Lengend Snippet: Fig. 7. Overexpression of CIP2A in astrocytes induces increased production of in- flammatory cytokines and Ab in mouse brains. (A) IL-1a, IL-6, and TNF-a in brain homogenates from the cortex and hippocampus of the mice were detected by ELISA, n ¼ 6 per group. (B) Ab40 and Ab42 levels in brain homogenates were detected by ELISA, n ¼ 6 per group. *** p < 0.001 versus vector. Data are mean SEM. (C) Cultured primary astrocytes were treated with Ab oligomers (2 mM) for 24 hours; CIP2A levels were detected by Western blotting. Left, representative blots. Right, Quantitative analysis of the protein levels, n ¼ 3 per group. *p < 0.05 versus con. Data are mean SEM. (D) Cell culture media in (C) were collected; IL-1a, IL-6, and TNF-a in culture media were detected by ELISA, n ¼ 9 well per group. * p < 0.05, *** p < 0.001,**** p < 0.0001 versus con. Data are mean SEM. (E) A working model: In AD brains, CIP2A is upregulated in astrocytes and induces reactive astrogliosis, the latter, results in over- production of toxic cytokines and Ab (identified in the present study), and probably reduced release of neurotrophins, thus promotes neuronal degeneration. Ab further stimulates CIP2A overexpression and thus forms a vicious cycle.

Article Snippet: Rabbit polyclonal antibody (pAb) against CIP2A (1:1000 for Western blotting) was from ABclonal (Boston, USA); another pAb of CIP2A (1:1000 for Western blotting and 1:200 for immunofluorescence) was from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Cell Culture, Western Blot