anti-gapdh Search Results


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  • 99
    Millipore anti gapdh antibody
    Antibody microarray analysis (a) Ratio distribution of differentially expressed proteins in ES cells treated with RA/AC compared with RA-treated samples. Of the 224 proteins represented in the array, 18 were upregulated (log 2 ratio ≥ 1) and 13 were downregulated (log 2 ratio ≤ 1). (b) STRING analysis of protein interactions. Nodes in red tones represent upregulated proteins and green tones represent downregulated proteins. Edges represent action types: blue = binding, red= inhibition, pink= postranslational modification, yellow=transcriptional regulation. (c) Representative immunoblot of <t>p-p38</t> and p-PI3K expression in EB samples treated with vehicle (control), RA, AC, or RA/AC. (d) Relative protein levels measured by Western blot and normalized to <t>GAPDH</t> levels. ** P ≤ 0.01; *** P ≤ 0.001.
    Anti Gapdh Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Santa Cruz Biotechnology anti gapdh antibody
    Effects of EGCG on the protein expression of <t>TGF-β1.</t> TGF-β1 and <t>GAPDH</t> expression was assessed by Western blotting with anti-TGF-β1 mAb or anti-GAPDH mAb, respectively. Results are shown in the top panels and quantified by densitometry (bottom panels). * p
    Anti Gapdh Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gapdh antibody/product/Santa Cruz Biotechnology
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    99
    Millipore anti gapdh monoclonal ab
    In vitro human RPE <t>GAPDH</t> and <t>nitrotyrosine.</t> Cultured human RPE cells (2 nd passage) were exposed to control medium (A,C) or medium containing 200 μM hydrogen peroxide (B,D) for 24 hours. Immumocytochemistry for GAPDH (A,B) and nitrotyrosine (C,D)
    Anti Gapdh Monoclonal Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    American Research Products anti gapdh antibody
    Levels of <t>ABCC4</t> in cells established using the Flp-In™ system. ABCC4 and <t>GAPDH</t> levels were determined by western blotting analysis with specific antibodies for ABCC4 and GAPDH and their levels were quantified using ImageJ (Wayne Rasband, Bethesda, MD, USA) as described in Materials and Methods. ABCC4-specific monoclonal antibody (M4I-10) or GAPDH-specific antibody was used for protein detection in PNGase F-treated cell lysate. The experiments were performed independently more than two times. Data are expressed as mean values ± S.D. (n = 3). Statistical analyses were performed using one-way ANOVA and Tukey’s HSD test (* p
    Anti Gapdh Antibody, supplied by American Research Products, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti gapdh antibody
    BACE1 overexpression reduces the surface levels of Na v ). Four matching slice pairs from similar locations of the brain were selected from BACE1-transgenic (BACE1-tg) and age-matched wild-type control mice (WT), respectively. Total lysate (1/20th of total input lysate, left panel ) and NeutrAvidin captured surface biotinylated proteins ( right panel ) were analyzed by Western blot (WB) using antibodies against Na v 1 <t>α-subunits</t> (pan) and control <t>GAPDH.</t>
    Anti Gapdh Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti gapdh antibody
    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on <t>JNK</t> phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; <t>GAPDH</t> antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p
    Anti Gapdh Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Trevigen anti gapdh ab
    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on <t>JNK</t> phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; <t>GAPDH</t> antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p
    Anti Gapdh Ab, supplied by Trevigen, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend anti gapdh primary ab
    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on <t>JNK</t> phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; <t>GAPDH</t> antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p
    Anti Gapdh Primary Ab, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Proteintech anti gapdh antibodies
    Effects of PCNP on the apoptosis of human neuroblastoma cells. a The apoptotic levels of SH-SY5Y and SK-N-SH cells were measured by TUNEL staining; original magnification 100×. b The percentages of TUNEL-positive cells were calculated by the formula shown above. c Western blotting analysis for the expression of cleaved <t>caspase-3,</t> − 8, and − 9 and cleaved PARP in SH-SY5Y and SK-N-SH cells. <t>GAPDH</t> was used as the loading control. ( d, e ) The densitometry analysis of each factor was performed in SH-SY5Y and SK-N-SH cells, normalized to the corresponding GAPDH level. Data are presented as mean ± SEM of three independent experiments; * P
    Anti Gapdh Antibodies, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Elabscience anti gapdh ab
    Effects of PCNP on the apoptosis of human neuroblastoma cells. a The apoptotic levels of SH-SY5Y and SK-N-SH cells were measured by TUNEL staining; original magnification 100×. b The percentages of TUNEL-positive cells were calculated by the formula shown above. c Western blotting analysis for the expression of cleaved <t>caspase-3,</t> − 8, and − 9 and cleaved PARP in SH-SY5Y and SK-N-SH cells. <t>GAPDH</t> was used as the loading control. ( d, e ) The densitometry analysis of each factor was performed in SH-SY5Y and SK-N-SH cells, normalized to the corresponding GAPDH level. Data are presented as mean ± SEM of three independent experiments; * P
    Anti Gapdh Ab, supplied by Elabscience, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti gapdh antibody
    miR-130b-3p participates in the regulation of Dsg1 expression. ( a ) Primary human keratinocytes were isolated as described in Materials and Methods and plated on dishes. Before they reached confluence, cells were induced to differentiate by adding 1.8 mM CaCl 2 to the culture medium. Cells were collected at the indicated time points to perform western blot analysis. Differentiation was evaluated by western blot for involucrin, <t>K10</t> and TG1 ( a ). Dsg1 protein ( b ) upregulation during differentiation is also shown; <t>GAPDH</t> is used as loading control. The histograms show the quantitative mRNA level of involucrin, K10, TG1 and Dsg1 (fold over control; black bars) as mean±S.D. from three independent experiments. ( c ) Keratinocytes were exposed to calcium stimulation and miR-130b-3p levels were analyzed by qRT-PCR. Data are shown as mean±S.D. from three independent experiments. ( d ) Putative miR-130b-3p-binding site in the 3’-UTR region of Dsg1. ( e ) miR-130b-3p suppresses the expression of Dsg1. Keratinocytes were transfected with mimic-miR-130b-3p (miR-130b-3p) or mimic negative control (mimic-NC). The levels of Dsg1 were analyzed by immunoblot. n =3. ( f ) Knockdown of miR-130b-3p increases the expression levels of Dsg1. Keratinocytes were transfected with miR-130b-3p antagomir (anta-130b-3p) or the antagomir-negative control (anta-NC), the levels of Dsg1 were analyzed by immunoblot; n =3. ( g ) Keratinocytes were treated with miR-130b-3p or mimic-NC, and cells were exposed to 1.8 mM CaCl 2 for 72 h. The levels of Dsg1 were analyzed by immunoblot. n =3. * P
    Anti Gapdh Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Charles River Laboratories anti gapdh polyclonal ab
    miR-130b-3p participates in the regulation of Dsg1 expression. ( a ) Primary human keratinocytes were isolated as described in Materials and Methods and plated on dishes. Before they reached confluence, cells were induced to differentiate by adding 1.8 mM CaCl 2 to the culture medium. Cells were collected at the indicated time points to perform western blot analysis. Differentiation was evaluated by western blot for involucrin, <t>K10</t> and TG1 ( a ). Dsg1 protein ( b ) upregulation during differentiation is also shown; <t>GAPDH</t> is used as loading control. The histograms show the quantitative mRNA level of involucrin, K10, TG1 and Dsg1 (fold over control; black bars) as mean±S.D. from three independent experiments. ( c ) Keratinocytes were exposed to calcium stimulation and miR-130b-3p levels were analyzed by qRT-PCR. Data are shown as mean±S.D. from three independent experiments. ( d ) Putative miR-130b-3p-binding site in the 3’-UTR region of Dsg1. ( e ) miR-130b-3p suppresses the expression of Dsg1. Keratinocytes were transfected with mimic-miR-130b-3p (miR-130b-3p) or mimic negative control (mimic-NC). The levels of Dsg1 were analyzed by immunoblot. n =3. ( f ) Knockdown of miR-130b-3p increases the expression levels of Dsg1. Keratinocytes were transfected with miR-130b-3p antagomir (anta-130b-3p) or the antagomir-negative control (anta-NC), the levels of Dsg1 were analyzed by immunoblot; n =3. ( g ) Keratinocytes were treated with miR-130b-3p or mimic-NC, and cells were exposed to 1.8 mM CaCl 2 for 72 h. The levels of Dsg1 were analyzed by immunoblot. n =3. * P
    Anti Gapdh Polyclonal Ab, supplied by Charles River Laboratories, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology rabbit anti gapdh antibody
    Regulation of miR-9 pri-miRNA expression by <t>TLX</t> a. RT-PCR analysis of miR-9 pri-miRNAs, miR-9-1 and miR-9-2, in wild type and TLX-null brains. <t>GAPDH</t> was included as a loading control. b. Schematics of miR-9-1 gene with consensus TLX binding sites (TLX-1/2, 3, 4) and REST responsive element (RE). The numbers are relative to miR-9 hairpin precursor ending site. The locations of the PCR primers for ChIP assays are indicated by arrows. c. ChIP assays to show binding of TLX to the consensus TLX binding sites downstream of miR-9-1 gene. d. ChIP assays to show binding of HDAC5 to TLX binding sites TLX-1/2 and TLX-3. The association of these sites with H3K9me3, but not with AcH3 and H3K4me3, was shown in the same assays. e. TLX represses miR-9-1 reporter activity. Luciferase activity of wild type (WT) or mutant (MT) miR-9-1 reporter gene was measured in the absence (-) or presence (+) of transfected TLX expression vector in mouse neural stem cells. Error bars are s.d. of the mean. * p=0.009 by Student's t-test.
    Rabbit Anti Gapdh Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Antibody microarray analysis (a) Ratio distribution of differentially expressed proteins in ES cells treated with RA/AC compared with RA-treated samples. Of the 224 proteins represented in the array, 18 were upregulated (log 2 ratio ≥ 1) and 13 were downregulated (log 2 ratio ≤ 1). (b) STRING analysis of protein interactions. Nodes in red tones represent upregulated proteins and green tones represent downregulated proteins. Edges represent action types: blue = binding, red= inhibition, pink= postranslational modification, yellow=transcriptional regulation. (c) Representative immunoblot of p-p38 and p-PI3K expression in EB samples treated with vehicle (control), RA, AC, or RA/AC. (d) Relative protein levels measured by Western blot and normalized to GAPDH levels. ** P ≤ 0.01; *** P ≤ 0.001.

    Journal: Differentiation; research in biological diversity

    Article Title: RARβ2-dependent signaling represses neuronal differentiation in mouse ES cells

    doi: 10.1016/j.diff.2017.11.002

    Figure Lengend Snippet: Antibody microarray analysis (a) Ratio distribution of differentially expressed proteins in ES cells treated with RA/AC compared with RA-treated samples. Of the 224 proteins represented in the array, 18 were upregulated (log 2 ratio ≥ 1) and 13 were downregulated (log 2 ratio ≤ 1). (b) STRING analysis of protein interactions. Nodes in red tones represent upregulated proteins and green tones represent downregulated proteins. Edges represent action types: blue = binding, red= inhibition, pink= postranslational modification, yellow=transcriptional regulation. (c) Representative immunoblot of p-p38 and p-PI3K expression in EB samples treated with vehicle (control), RA, AC, or RA/AC. (d) Relative protein levels measured by Western blot and normalized to GAPDH levels. ** P ≤ 0.01; *** P ≤ 0.001.

    Article Snippet: The blots were probed with anti-GAPDH (SAB2108266, 1:500, Sigma-Aldrich), anti-phospho-p38 MAPK (44-684G, 1:1000, Invitrogen/Thermo Fisher Scientific), and anti-phospho-PI3K (4292, 1:1000, Cell Signaling Technology).

    Techniques: Microarray, Binding Assay, Inhibition, Modification, Expressing, Western Blot

    ( A ) The immunoblots of PKCζ, Survivin, MMP2, MMP9, and GAPDH in HeLa cells. ( B ) The immunoblots of p-STAT3 and STAT3 in HeLa cells. ( C ) The immunoblots of STAT3 and Histone H3 in nuclei of HeLa cells. ( D ) Columns indicated the relative expression levels of PKCζ, Survivin, MMP2, MMP9, and GAPDH in HeLa cells. ( E ) Columns indicated the phosphorylation level of STAT3 in HeLa cells. ( F ) Columns indicated the relative expression level of STAT3 in nuclei of HeLa cells. ( G ) The immunoblots of PKCζ, Survivin, MMP2, MMP9, and GAPDH in SiHa cells. ( H ) The immunoblots of p-STAT3 and STAT3 in SiHa cells. ( I ) The immunoblots of STAT3 and Histone H3 in nuclei of SiHa cells. ( J ) Columns indicated the relative expression levels of PKCζ, Survivin, MMP2, MMP9, and GAPDH in SiHa cells. ( K ) Columns indicated the phosphorylation level of STAT3 in SiHa cells. ( L ) Columns indicated the relative expression level of STAT3 in nuclei of SiHa cells.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Baicalin Inhibits Human Cervical Cancer Cells by Suppressing Protein Kinase C/Signal Transducer and Activator of Transcription (PKC/STAT3) Signaling Pathway

    doi: 10.12659/MSM.909640

    Figure Lengend Snippet: ( A ) The immunoblots of PKCζ, Survivin, MMP2, MMP9, and GAPDH in HeLa cells. ( B ) The immunoblots of p-STAT3 and STAT3 in HeLa cells. ( C ) The immunoblots of STAT3 and Histone H3 in nuclei of HeLa cells. ( D ) Columns indicated the relative expression levels of PKCζ, Survivin, MMP2, MMP9, and GAPDH in HeLa cells. ( E ) Columns indicated the phosphorylation level of STAT3 in HeLa cells. ( F ) Columns indicated the relative expression level of STAT3 in nuclei of HeLa cells. ( G ) The immunoblots of PKCζ, Survivin, MMP2, MMP9, and GAPDH in SiHa cells. ( H ) The immunoblots of p-STAT3 and STAT3 in SiHa cells. ( I ) The immunoblots of STAT3 and Histone H3 in nuclei of SiHa cells. ( J ) Columns indicated the relative expression levels of PKCζ, Survivin, MMP2, MMP9, and GAPDH in SiHa cells. ( K ) Columns indicated the phosphorylation level of STAT3 in SiHa cells. ( L ) Columns indicated the relative expression level of STAT3 in nuclei of SiHa cells.

    Article Snippet: Agents and antibodies Agents and antibodies included: baicalin (Sigma-Aldrich, Cat# 572667), TUNEL kit (Roche, Cat# 11684795910), PKCζ antibody (Cell Signaling Tech, Cat#9372, 1: 4000), STAT3 antibody (Cell Signaling Tech, Cat#4368, 1: 2000), phosphorylated STAT3 antibody (p-STAT3, Cell Signaling Tech, Cat#8119, 1: 2000), Survivin antibody (Abcam, Cat#ab76424, 1: 4000), matrix metalloproteinase (MMP)2 antibody (Abcam, Cat#ab37150, 1: 4000), MMP9 antibody (Abcam, Cat#ab38898, 1: 4000), Histone H3 antibody (Abcam, Cat#ab8580, 1: 4000), and GAPDH antibody (Sigma-Aldrich, Cat#G9545, 1: 6000).

    Techniques: Western Blot, Expressing

    Effects of EGCG on the protein expression of TGF-β1. TGF-β1 and GAPDH expression was assessed by Western blotting with anti-TGF-β1 mAb or anti-GAPDH mAb, respectively. Results are shown in the top panels and quantified by densitometry (bottom panels). * p

    Journal: Chonnam Medical Journal

    Article Title: Effects of Epigallocatechin-3-Gallate on the Expression of TGF-?1, PKC ?/?II, and NF-?B in High-Glucose-Stimulated Glomerular Epithelial Cells

    doi: 10.4068/cmj.2011.47.2.116

    Figure Lengend Snippet: Effects of EGCG on the protein expression of TGF-β1. TGF-β1 and GAPDH expression was assessed by Western blotting with anti-TGF-β1 mAb or anti-GAPDH mAb, respectively. Results are shown in the top panels and quantified by densitometry (bottom panels). * p

    Article Snippet: Anti-phospho-PKC α/βII (Thr638/641) antibody was purchased from Cell Signaling Technologies Inc. (Beverly, MA), and anti-TGF-β1 antibody, anti-PKC-α antibody, and anti-GAPDH antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Expressing, Western Blot

    Effects of EGCG on the mRNA expression of TGF-β1. (A) GECs were pretreated with EGCG (100 µM), glucose (30 mM), or a combination of the two for 24 hours. Cellular RNAs were extracted and the mRNA expression of TGF-β1 and GAPDH was analyzed by RT-PCR. (B) The mRNA expression of TGF-β1 was measured by real-time RT-PCR as described in Materials and Methods. * p

    Journal: Chonnam Medical Journal

    Article Title: Effects of Epigallocatechin-3-Gallate on the Expression of TGF-?1, PKC ?/?II, and NF-?B in High-Glucose-Stimulated Glomerular Epithelial Cells

    doi: 10.4068/cmj.2011.47.2.116

    Figure Lengend Snippet: Effects of EGCG on the mRNA expression of TGF-β1. (A) GECs were pretreated with EGCG (100 µM), glucose (30 mM), or a combination of the two for 24 hours. Cellular RNAs were extracted and the mRNA expression of TGF-β1 and GAPDH was analyzed by RT-PCR. (B) The mRNA expression of TGF-β1 was measured by real-time RT-PCR as described in Materials and Methods. * p

    Article Snippet: Anti-phospho-PKC α/βII (Thr638/641) antibody was purchased from Cell Signaling Technologies Inc. (Beverly, MA), and anti-TGF-β1 antibody, anti-PKC-α antibody, and anti-GAPDH antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Slit2-N inhibited the LPS-induced cytokine and cell adhesion molecule ICAM-1 expression in HUVECs as well as monocyte adhesion on HUVECs HUVECs were treated with 100ng/mL LPS for 2 h (A) or 12 h (B) with or without treatment of Slit2-N. (A) Cells were stimulated by LPS with pre-treatment of a gradient of 3 nmol/L, 30 nmol/L and 60 nmol/L Slit2-N or post-treatment with 30 nmol/L Slit2-N (post-Slit2-N). Total RNA was isolated and qRT-PCR was performed to detect mRNA expression level of MCP-1 and GM-CSF. Values are normalized to control group, and are not comparable between cytokines. (B) Cell culture supernatants were collected after Slit2-N (30 nmol/L) plus LPS treatment. Cytokines secreted in the supernatants were detected with R D cytokine array kit. Values are net optical signaling intensity. (C) HUVECs were treated with 100 ng/mL LPS for 12 h with or without pre-treatment of Slit2-N (30 nmol/L). Cells were lysed and WB was performed using ICAM-1, VCAM-1 and GAPDH antibodies. (D) HUVECs were stimulated with LPS (100 ng/mL) for 6 h with or without Slit2-N (30 nmol/L). CFSE labeled THP-1 monocytic cells were added onto treated HUVEC. And after washing, attached THP-1 cells were quantified by mean fluorescence intensity at 528nm. (E) HMVECs were stimulated with 100ng/mL LPS in the presence or absence of 30 nmol/L Slit2-N for 12 h. Supernatant MCP-1 secretion was analyzed by ELISA. (F) ICAM-1 expression was detected by qRT-PCR in HMVECs with LPS stimulation in the presence or absence of Slit2-N. (* p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Slit2-Robo4 pathway modulates LPS-induced endothelial inflammation and its expression is dysregulated during endotoxemia

    doi: 10.4049/jimmunol.1302021

    Figure Lengend Snippet: Slit2-N inhibited the LPS-induced cytokine and cell adhesion molecule ICAM-1 expression in HUVECs as well as monocyte adhesion on HUVECs HUVECs were treated with 100ng/mL LPS for 2 h (A) or 12 h (B) with or without treatment of Slit2-N. (A) Cells were stimulated by LPS with pre-treatment of a gradient of 3 nmol/L, 30 nmol/L and 60 nmol/L Slit2-N or post-treatment with 30 nmol/L Slit2-N (post-Slit2-N). Total RNA was isolated and qRT-PCR was performed to detect mRNA expression level of MCP-1 and GM-CSF. Values are normalized to control group, and are not comparable between cytokines. (B) Cell culture supernatants were collected after Slit2-N (30 nmol/L) plus LPS treatment. Cytokines secreted in the supernatants were detected with R D cytokine array kit. Values are net optical signaling intensity. (C) HUVECs were treated with 100 ng/mL LPS for 12 h with or without pre-treatment of Slit2-N (30 nmol/L). Cells were lysed and WB was performed using ICAM-1, VCAM-1 and GAPDH antibodies. (D) HUVECs were stimulated with LPS (100 ng/mL) for 6 h with or without Slit2-N (30 nmol/L). CFSE labeled THP-1 monocytic cells were added onto treated HUVEC. And after washing, attached THP-1 cells were quantified by mean fluorescence intensity at 528nm. (E) HMVECs were stimulated with 100ng/mL LPS in the presence or absence of 30 nmol/L Slit2-N for 12 h. Supernatant MCP-1 secretion was analyzed by ELISA. (F) ICAM-1 expression was detected by qRT-PCR in HMVECs with LPS stimulation in the presence or absence of Slit2-N. (* p

    Article Snippet: ICAM-1, VCAM-1 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Cell Culture, Western Blot, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay

    In vitro human RPE GAPDH and nitrotyrosine. Cultured human RPE cells (2 nd passage) were exposed to control medium (A,C) or medium containing 200 μM hydrogen peroxide (B,D) for 24 hours. Immumocytochemistry for GAPDH (A,B) and nitrotyrosine (C,D)

    Journal: Redox report : communications in free radical research

    Article Title: Ischemia-induced nitrotyrosine formation and nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in human retinal pigment epithelium in vivo

    doi: 10.1179/174329211X12968219310710

    Figure Lengend Snippet: In vitro human RPE GAPDH and nitrotyrosine. Cultured human RPE cells (2 nd passage) were exposed to control medium (A,C) or medium containing 200 μM hydrogen peroxide (B,D) for 24 hours. Immumocytochemistry for GAPDH (A,B) and nitrotyrosine (C,D)

    Article Snippet: After blocking procedures, sections were incubated with 2 μg/mL mouse monoclonal anti-nitrotyrosine antibody (1:50; Millipore, Billerica, MA), monoclonal anti-GAPDH antibody (1:50; Millipore) or isotype control IgG diluted in 1% BSA in PBS overnight at 4°C.

    Techniques: In Vitro, Cell Culture

    In vivo human RPE and choroidal GAPDH and nitrotyrosine. Ischemic choroid and RPE (A) demonstrate GAPDH nuclear translocation in RPE and vascular endothelial cells (C) as well as considerable immunoreactive nitrotyrosine (E). RPE and choroid of control

    Journal: Redox report : communications in free radical research

    Article Title: Ischemia-induced nitrotyrosine formation and nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in human retinal pigment epithelium in vivo

    doi: 10.1179/174329211X12968219310710

    Figure Lengend Snippet: In vivo human RPE and choroidal GAPDH and nitrotyrosine. Ischemic choroid and RPE (A) demonstrate GAPDH nuclear translocation in RPE and vascular endothelial cells (C) as well as considerable immunoreactive nitrotyrosine (E). RPE and choroid of control

    Article Snippet: After blocking procedures, sections were incubated with 2 μg/mL mouse monoclonal anti-nitrotyrosine antibody (1:50; Millipore, Billerica, MA), monoclonal anti-GAPDH antibody (1:50; Millipore) or isotype control IgG diluted in 1% BSA in PBS overnight at 4°C.

    Techniques: In Vivo, Translocation Assay

    Generation and characterization of apoE-fKI/Dlx-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Dlx-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–F , ZsGreen1 was expressed in GABA-positive ( C , E ) and somatostatin (SOM)-positive ( D , F ) inhibitory interneurons in the cortex and hippocampus of Dlx-Cre-positive apoE-fKI mice. Scale bars: 15 μm. G , H , Anti-apoE immunostaining revealed the presence of apoE protein in GABA-positive hilar interneurons in aged apoE4-fKI mice ( G ), but not in apoE4-fKI/Dlx-Cre mice ( H ). Scale bars: 15 μm. I , Images of GABA-positive hilar interneurons before and after laser capture. Inhibitory interneuron were identified by anti-GABA immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 15 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Dlx-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Dlx-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). For both the cortex and the hippocampus, the apoE level in apoE3-fKI mice was normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p

    Journal: The Journal of Neuroscience

    Article Title: Apolipoprotein E4 Produced in GABAergic Interneurons Causes Learning and Memory Deficits in Mice

    doi: 10.1523/JNEUROSCI.2281-14.2014

    Figure Lengend Snippet: Generation and characterization of apoE-fKI/Dlx-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Dlx-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–F , ZsGreen1 was expressed in GABA-positive ( C , E ) and somatostatin (SOM)-positive ( D , F ) inhibitory interneurons in the cortex and hippocampus of Dlx-Cre-positive apoE-fKI mice. Scale bars: 15 μm. G , H , Anti-apoE immunostaining revealed the presence of apoE protein in GABA-positive hilar interneurons in aged apoE4-fKI mice ( G ), but not in apoE4-fKI/Dlx-Cre mice ( H ). Scale bars: 15 μm. I , Images of GABA-positive hilar interneurons before and after laser capture. Inhibitory interneuron were identified by anti-GABA immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 15 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Dlx-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Dlx-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). For both the cortex and the hippocampus, the apoE level in apoE3-fKI mice was normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p

    Article Snippet: Nonspecific binding sites were blocked with blocking buffer (LI-COR Biosciences) for 1 h. Membranes were incubated with polyclonal goat anti-apoE antibody (1:5000; Calbiochem) and monoclonal mouse anti-GAPDH antibody (1:10,000; Millipore Bioscience Research Reagents) overnight at 4°C.

    Techniques: Mouse Assay, Expressing, Immunostaining, Binding Assay, Polymerase Chain Reaction, Amplification, Western Blot

    Generation and characterization of apoE-fKI/Syn-1-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Syn-1-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–H , ZsGreen1 was expressed in NeuN-positive neurons ( C , F ) and GABA-positive inhibitory interneurons ( E , H ), but not in GFAP-positive astrocytes ( D , G ) in the cortex and hippocampus of Syn-1-Cre-positive apoE-fKI mice. Scale bars: 30 μm. I , Images of the dentate granular cell layer before and after laser capture. Neuronal nuclei were identified by anti-NeuN immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 30 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Syn-1-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Syn-1-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p

    Journal: The Journal of Neuroscience

    Article Title: Apolipoprotein E4 Produced in GABAergic Interneurons Causes Learning and Memory Deficits in Mice

    doi: 10.1523/JNEUROSCI.2281-14.2014

    Figure Lengend Snippet: Generation and characterization of apoE-fKI/Syn-1-Cre mice. A , B , Expression of ZsGreen1 (green) in the cortex and hippocampus of Syn-1-Cre-positive ( A ) and -negative ( B ) ZsGreen1 reporter mice. Scale bars: 250 μm. C–H , ZsGreen1 was expressed in NeuN-positive neurons ( C , F ) and GABA-positive inhibitory interneurons ( E , H ), but not in GFAP-positive astrocytes ( D , G ) in the cortex and hippocampus of Syn-1-Cre-positive apoE-fKI mice. Scale bars: 30 μm. I , Images of the dentate granular cell layer before and after laser capture. Neuronal nuclei were identified by anti-NeuN immunostaining. Arrows indicate the cell before and after laser capture. Scale bars: 30 μm. J , Schematic of primers and their binding sites on the human APOE gene. K , PCR with primers 1 and 2 resulted in an amplified product in samples of apoE4-fKI mice, but not in samples of apoE4-fKI/Syn-1-Cre mice (left). PCR with primers 1 and 3 resulted in an amplified product in samples of apoE4-fKI/Syn-1-Cre mice, but not in samples of apoE4-fKI mice (right). Fifty nuclei per sample were used. L , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. M , N , Quantification of apoE protein levels relative to GAPDH protein levels in cortical ( M ) and hippocampal lysates ( N ) of 17-month-old mice ( n = 5 per genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. * p

    Article Snippet: Nonspecific binding sites were blocked with blocking buffer (LI-COR Biosciences) for 1 h. Membranes were incubated with polyclonal goat anti-apoE antibody (1:5000; Calbiochem) and monoclonal mouse anti-GAPDH antibody (1:10,000; Millipore Bioscience Research Reagents) overnight at 4°C.

    Techniques: Mouse Assay, Expressing, Immunostaining, Binding Assay, Polymerase Chain Reaction, Amplification, Western Blot

    Generation and characterization of apoE-fKI/GFAP-Cre mice. A , B , Representative images of fluorescent immunostaining with anti-Cre recombinase (green) and anti-GFAP (red) in the cortex ( A ) and hippocampus ( B ) of apoE-fKI/GFAP-Cre mice. C , D , Neurons immunostained with anti-NeuN (red) did not express Cre-recombinase in the cortex ( C ) or hippocampus ( D ) of apoE-fKI/GFAP-Cre mice. E , F , Anti-apoE (green) and anti-GFAP (red) double immunostaining revealed that apoE expression was dramatically decreased in cortical ( E ) and hippocampal ( F ) astrocytes in apoE3-fKI/GFAP-Cre and apoE4-fKI/GFAP-Cre mice. G , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. H , I , Quantification of apoE protein levels relative to GAPDH in cortical ( H ) and hippocampal lysates ( I ) of 17-month-old mice ( n = 5/genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. *** p

    Journal: The Journal of Neuroscience

    Article Title: Apolipoprotein E4 Produced in GABAergic Interneurons Causes Learning and Memory Deficits in Mice

    doi: 10.1523/JNEUROSCI.2281-14.2014

    Figure Lengend Snippet: Generation and characterization of apoE-fKI/GFAP-Cre mice. A , B , Representative images of fluorescent immunostaining with anti-Cre recombinase (green) and anti-GFAP (red) in the cortex ( A ) and hippocampus ( B ) of apoE-fKI/GFAP-Cre mice. C , D , Neurons immunostained with anti-NeuN (red) did not express Cre-recombinase in the cortex ( C ) or hippocampus ( D ) of apoE-fKI/GFAP-Cre mice. E , F , Anti-apoE (green) and anti-GFAP (red) double immunostaining revealed that apoE expression was dramatically decreased in cortical ( E ) and hippocampal ( F ) astrocytes in apoE3-fKI/GFAP-Cre and apoE4-fKI/GFAP-Cre mice. G , Representative fluorescent Western blot of apoE (green) and GAPDH (red) in cortical and hippocampal lysates of 17-month-old female mice with different genotypes. H , I , Quantification of apoE protein levels relative to GAPDH in cortical ( H ) and hippocampal lysates ( I ) of 17-month-old mice ( n = 5/genotype). ApoE levels in apoE3-fKI mice were normalized to 1, and apoE levels in other groups of mice were presented relative to those in apoE3-fKI mice. *** p

    Article Snippet: Nonspecific binding sites were blocked with blocking buffer (LI-COR Biosciences) for 1 h. Membranes were incubated with polyclonal goat anti-apoE antibody (1:5000; Calbiochem) and monoclonal mouse anti-GAPDH antibody (1:10,000; Millipore Bioscience Research Reagents) overnight at 4°C.

    Techniques: Mouse Assay, Immunostaining, Double Immunostaining, Expressing, Western Blot

    Levels of ABCC4 in cells established using the Flp-In™ system. ABCC4 and GAPDH levels were determined by western blotting analysis with specific antibodies for ABCC4 and GAPDH and their levels were quantified using ImageJ (Wayne Rasband, Bethesda, MD, USA) as described in Materials and Methods. ABCC4-specific monoclonal antibody (M4I-10) or GAPDH-specific antibody was used for protein detection in PNGase F-treated cell lysate. The experiments were performed independently more than two times. Data are expressed as mean values ± S.D. (n = 3). Statistical analyses were performed using one-way ANOVA and Tukey’s HSD test (* p

    Journal: Cells

    Article Title: A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance

    doi: 10.3390/cells8010039

    Figure Lengend Snippet: Levels of ABCC4 in cells established using the Flp-In™ system. ABCC4 and GAPDH levels were determined by western blotting analysis with specific antibodies for ABCC4 and GAPDH and their levels were quantified using ImageJ (Wayne Rasband, Bethesda, MD, USA) as described in Materials and Methods. ABCC4-specific monoclonal antibody (M4I-10) or GAPDH-specific antibody was used for protein detection in PNGase F-treated cell lysate. The experiments were performed independently more than two times. Data are expressed as mean values ± S.D. (n = 3). Statistical analyses were performed using one-way ANOVA and Tukey’s HSD test (* p

    Article Snippet: The membranes were incubated with monoclonal anti-ABCC4 antibody (M4I-10; GeneTex Inc., Alton Parkway Irvine, CA, USA) or anti-GAPDH antibody (anti-GAPDH-Clone 6C5 mouse monoclonal, igG2b; American Research Products, Inc., Waltham, MA, USA) at 1:1000 dilution in TBST containing 5% (w /v ) skim milk powder for 1 h at room temperature with shaking after rinsing with TBST.

    Techniques: Western Blot

    Levels of ABCC4 mRNA in cells established using the Flp-In™ system. The levels of ABCC4 and GAPDH mRNA were measured using qPCR with specific primer sets for ABCC4 and GAPDH , as described in Materials and Methods. Data are calculated as ratios relative to the GAPDH mRNA levels in the cells and normalized to the ratio of ABCC4 / GAPDH . Data are expressed as mean values ± S.D. (n = 5). Statistical analyses were performed using one-way ANOVA and Tukey’s HSD test (* p

    Journal: Cells

    Article Title: A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance

    doi: 10.3390/cells8010039

    Figure Lengend Snippet: Levels of ABCC4 mRNA in cells established using the Flp-In™ system. The levels of ABCC4 and GAPDH mRNA were measured using qPCR with specific primer sets for ABCC4 and GAPDH , as described in Materials and Methods. Data are calculated as ratios relative to the GAPDH mRNA levels in the cells and normalized to the ratio of ABCC4 / GAPDH . Data are expressed as mean values ± S.D. (n = 5). Statistical analyses were performed using one-way ANOVA and Tukey’s HSD test (* p

    Article Snippet: The membranes were incubated with monoclonal anti-ABCC4 antibody (M4I-10; GeneTex Inc., Alton Parkway Irvine, CA, USA) or anti-GAPDH antibody (anti-GAPDH-Clone 6C5 mouse monoclonal, igG2b; American Research Products, Inc., Waltham, MA, USA) at 1:1000 dilution in TBST containing 5% (w /v ) skim milk powder for 1 h at room temperature with shaking after rinsing with TBST.

    Techniques: Real-time Polymerase Chain Reaction

    BACE1 overexpression reduces the surface levels of Na v ). Four matching slice pairs from similar locations of the brain were selected from BACE1-transgenic (BACE1-tg) and age-matched wild-type control mice (WT), respectively. Total lysate (1/20th of total input lysate, left panel ) and NeutrAvidin captured surface biotinylated proteins ( right panel ) were analyzed by Western blot (WB) using antibodies against Na v 1 α-subunits (pan) and control GAPDH.

    Journal: Methods in Molecular Biology (Clifton, N.j.)

    Article Title: Surface Trafficking of Sodium Channels in Cells and in Hippocampal Slices

    doi: 10.1007/978-1-61779-328-8_23

    Figure Lengend Snippet: BACE1 overexpression reduces the surface levels of Na v ). Four matching slice pairs from similar locations of the brain were selected from BACE1-transgenic (BACE1-tg) and age-matched wild-type control mice (WT), respectively. Total lysate (1/20th of total input lysate, left panel ) and NeutrAvidin captured surface biotinylated proteins ( right panel ) were analyzed by Western blot (WB) using antibodies against Na v 1 α-subunits (pan) and control GAPDH.

    Article Snippet: Primary antibody solutions used in our study: Anti-pan α-subunit antibody (1:500, Sigma), anti-Nav 1.1 α-subunit antibody (1:500, Sigma), anti-Nav 1.2 α-subunit antibody (1:500, Alomone, Isael), anti-GAPDH antibody (BD bioscience), anti- N -cadherin antibody (BD bioscience/transduction laboratory).

    Techniques: Over Expression, Transgenic Assay, Mouse Assay, Western Blot

    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Journal: Neurochemistry International

    Article Title: Effect of Glutamate and Riluzole on Manganese-Induced Apoptotic Cell Signaling in Neuronally Differentiated Mouse P19 Cells

    doi: 10.1016/j.neuint.2012.04.015

    Figure Lengend Snippet: Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Article Snippet: Phospho-JNK antibody, anti-GAPDH antibody and secondary antibody for Western blots were obtained from Cell Signaling (Danvers, MA) and Western lightning plus substrate to develop immunoblots was from Perkin Elmer (Waltham, MA).

    Techniques: Western Blot

    Effects of PCNP on the apoptosis of human neuroblastoma cells. a The apoptotic levels of SH-SY5Y and SK-N-SH cells were measured by TUNEL staining; original magnification 100×. b The percentages of TUNEL-positive cells were calculated by the formula shown above. c Western blotting analysis for the expression of cleaved caspase-3, − 8, and − 9 and cleaved PARP in SH-SY5Y and SK-N-SH cells. GAPDH was used as the loading control. ( d, e ) The densitometry analysis of each factor was performed in SH-SY5Y and SK-N-SH cells, normalized to the corresponding GAPDH level. Data are presented as mean ± SEM of three independent experiments; * P

    Journal: BMC Cancer

    Article Title: PEST-containing nuclear protein mediates the proliferation, migration, and invasion of human neuroblastoma cells through MAPK and PI3K/AKT/mTOR signaling pathways

    doi: 10.1186/s12885-018-4391-9

    Figure Lengend Snippet: Effects of PCNP on the apoptosis of human neuroblastoma cells. a The apoptotic levels of SH-SY5Y and SK-N-SH cells were measured by TUNEL staining; original magnification 100×. b The percentages of TUNEL-positive cells were calculated by the formula shown above. c Western blotting analysis for the expression of cleaved caspase-3, − 8, and − 9 and cleaved PARP in SH-SY5Y and SK-N-SH cells. GAPDH was used as the loading control. ( d, e ) The densitometry analysis of each factor was performed in SH-SY5Y and SK-N-SH cells, normalized to the corresponding GAPDH level. Data are presented as mean ± SEM of three independent experiments; * P

    Article Snippet: Anti-PCNP, Anti-B-cell lymphoma-2 (Bcl-2), anti-Bcl-2-associated X protein (Bax), anti-B-cell lymphoma-extra large (Bcl-xl), anti-Bcl-xl/Bcl-2-associated death promoter (Bad), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-Cleaved poly adenosine diphosphate-ribose polymerase (PARP), and anti-GAPDH antibodies were purchased from ProteinTech (Chicago, IL, USA).

    Techniques: TUNEL Assay, Staining, Western Blot, Expressing

    miR-130b-3p participates in the regulation of Dsg1 expression. ( a ) Primary human keratinocytes were isolated as described in Materials and Methods and plated on dishes. Before they reached confluence, cells were induced to differentiate by adding 1.8 mM CaCl 2 to the culture medium. Cells were collected at the indicated time points to perform western blot analysis. Differentiation was evaluated by western blot for involucrin, K10 and TG1 ( a ). Dsg1 protein ( b ) upregulation during differentiation is also shown; GAPDH is used as loading control. The histograms show the quantitative mRNA level of involucrin, K10, TG1 and Dsg1 (fold over control; black bars) as mean±S.D. from three independent experiments. ( c ) Keratinocytes were exposed to calcium stimulation and miR-130b-3p levels were analyzed by qRT-PCR. Data are shown as mean±S.D. from three independent experiments. ( d ) Putative miR-130b-3p-binding site in the 3’-UTR region of Dsg1. ( e ) miR-130b-3p suppresses the expression of Dsg1. Keratinocytes were transfected with mimic-miR-130b-3p (miR-130b-3p) or mimic negative control (mimic-NC). The levels of Dsg1 were analyzed by immunoblot. n =3. ( f ) Knockdown of miR-130b-3p increases the expression levels of Dsg1. Keratinocytes were transfected with miR-130b-3p antagomir (anta-130b-3p) or the antagomir-negative control (anta-NC), the levels of Dsg1 were analyzed by immunoblot; n =3. ( g ) Keratinocytes were treated with miR-130b-3p or mimic-NC, and cells were exposed to 1.8 mM CaCl 2 for 72 h. The levels of Dsg1 were analyzed by immunoblot. n =3. * P

    Journal: Cell Death & Disease

    Article Title: H19 lncRNA regulates keratinocyte differentiation by targeting miR-130b-3p

    doi: 10.1038/cddis.2017.516

    Figure Lengend Snippet: miR-130b-3p participates in the regulation of Dsg1 expression. ( a ) Primary human keratinocytes were isolated as described in Materials and Methods and plated on dishes. Before they reached confluence, cells were induced to differentiate by adding 1.8 mM CaCl 2 to the culture medium. Cells were collected at the indicated time points to perform western blot analysis. Differentiation was evaluated by western blot for involucrin, K10 and TG1 ( a ). Dsg1 protein ( b ) upregulation during differentiation is also shown; GAPDH is used as loading control. The histograms show the quantitative mRNA level of involucrin, K10, TG1 and Dsg1 (fold over control; black bars) as mean±S.D. from three independent experiments. ( c ) Keratinocytes were exposed to calcium stimulation and miR-130b-3p levels were analyzed by qRT-PCR. Data are shown as mean±S.D. from three independent experiments. ( d ) Putative miR-130b-3p-binding site in the 3’-UTR region of Dsg1. ( e ) miR-130b-3p suppresses the expression of Dsg1. Keratinocytes were transfected with mimic-miR-130b-3p (miR-130b-3p) or mimic negative control (mimic-NC). The levels of Dsg1 were analyzed by immunoblot. n =3. ( f ) Knockdown of miR-130b-3p increases the expression levels of Dsg1. Keratinocytes were transfected with miR-130b-3p antagomir (anta-130b-3p) or the antagomir-negative control (anta-NC), the levels of Dsg1 were analyzed by immunoblot; n =3. ( g ) Keratinocytes were treated with miR-130b-3p or mimic-NC, and cells were exposed to 1.8 mM CaCl 2 for 72 h. The levels of Dsg1 were analyzed by immunoblot. n =3. * P

    Article Snippet: The anti-Dsg1 antibody (1:500, Abcam, Cambridge, MA, USA), anti-involucrin antibody (1:300, Abcam), anti-K10 antibody (1:500, Abcam), anti-TG1 (1:200, Santa Cruz Biotechnology CA, USA) and anti-GAPDH antibody (1:2,000, Abcam) were used in this study.

    Techniques: Expressing, Isolation, Western Blot, Quantitative RT-PCR, Binding Assay, Transfection, Negative Control

    Trends of actinomycin V on p53, p21 Waf1/Cip1 and Bax expression. ( A ) Expressions of relative proteins were measured by Western blotting; ( B ) Relative mRNA expression values were calculated by real-time PCR after 1 nmol/L actinomycin V treatment. The quantity of each mRNA was relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. *** p

    Journal: Marine Drugs

    Article Title: Actinomycin V Suppresses Human Non-Small-Cell Lung Carcinoma A549 Cells by Inducing G2/M Phase Arrest and Apoptosis via the p53-Dependent Pathway

    doi: 10.3390/md17100572

    Figure Lengend Snippet: Trends of actinomycin V on p53, p21 Waf1/Cip1 and Bax expression. ( A ) Expressions of relative proteins were measured by Western blotting; ( B ) Relative mRNA expression values were calculated by real-time PCR after 1 nmol/L actinomycin V treatment. The quantity of each mRNA was relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. *** p

    Article Snippet: Bcl-2, Bax and GAPDH antibodies were supplied by Abcam, Inc. (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Regulation of miR-9 pri-miRNA expression by TLX a. RT-PCR analysis of miR-9 pri-miRNAs, miR-9-1 and miR-9-2, in wild type and TLX-null brains. GAPDH was included as a loading control. b. Schematics of miR-9-1 gene with consensus TLX binding sites (TLX-1/2, 3, 4) and REST responsive element (RE). The numbers are relative to miR-9 hairpin precursor ending site. The locations of the PCR primers for ChIP assays are indicated by arrows. c. ChIP assays to show binding of TLX to the consensus TLX binding sites downstream of miR-9-1 gene. d. ChIP assays to show binding of HDAC5 to TLX binding sites TLX-1/2 and TLX-3. The association of these sites with H3K9me3, but not with AcH3 and H3K4me3, was shown in the same assays. e. TLX represses miR-9-1 reporter activity. Luciferase activity of wild type (WT) or mutant (MT) miR-9-1 reporter gene was measured in the absence (-) or presence (+) of transfected TLX expression vector in mouse neural stem cells. Error bars are s.d. of the mean. * p=0.009 by Student's t-test.

    Journal: Nature structural & molecular biology

    Article Title: A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

    doi: 10.1038/nsmb.1576

    Figure Lengend Snippet: Regulation of miR-9 pri-miRNA expression by TLX a. RT-PCR analysis of miR-9 pri-miRNAs, miR-9-1 and miR-9-2, in wild type and TLX-null brains. GAPDH was included as a loading control. b. Schematics of miR-9-1 gene with consensus TLX binding sites (TLX-1/2, 3, 4) and REST responsive element (RE). The numbers are relative to miR-9 hairpin precursor ending site. The locations of the PCR primers for ChIP assays are indicated by arrows. c. ChIP assays to show binding of TLX to the consensus TLX binding sites downstream of miR-9-1 gene. d. ChIP assays to show binding of HDAC5 to TLX binding sites TLX-1/2 and TLX-3. The association of these sites with H3K9me3, but not with AcH3 and H3K4me3, was shown in the same assays. e. TLX represses miR-9-1 reporter activity. Luciferase activity of wild type (WT) or mutant (MT) miR-9-1 reporter gene was measured in the absence (-) or presence (+) of transfected TLX expression vector in mouse neural stem cells. Error bars are s.d. of the mean. * p=0.009 by Student's t-test.

    Article Snippet: The mutant miR-9 RNA duplex sense sequence is UGA AAC CAA AUC UAG CUG UAU GA. Western blot of TLX and GAPDH was performed using rabbit anti-TLX antibody (1:1,000) and rabbit anti-GAPDH antibody (Santa Cruz, 1:1,000).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Activity Assay, Luciferase, Mutagenesis, Transfection, Plasmid Preparation

    miR-9-directed repression of TLX expression a. miR-9 expression in adult mouse neural stem cells during a differentiation time course. Day 0 (d0) represents the undifferentiated neural stem cell state. U6 was included as a loading control. Relative miR-9 levels, normalized to U6 levels, are indicated under the blots, with miR-9 level in d0 designated as 1. b. Western blot analysis of TLX expression in the same differentiation time course. GAPDH was included as a loading control. c. miR-9-mediated repression of luciferase reporter gene upstream of TLX 3′ UTR. The TLX 3′ UTR reporter gene was co-transfected with increasing amounts of miR-9 RNA duplexes or control siRNA duplexes into HEK 293 cells. d. Similar reporter assays were performed in cells transfected with MDH1-PGK-GFP control vector (1) or miR-9-1 expression vector (2). e. Mutation of the miR-9 target site in TLX 3′ UTR abolished miR-9-mediated repression. Luciferase reporter gene under the control of wild type (WT) or mutant (Mut) TLX 3′ UTR was transfected along with control siRNA (C), a miR-9 mutant with mutations in the seed region complementary to the mutant TLX 3′ UTR, or wild type miR-9 into HEK 293 cells. s.d. is indicated by error bars. f, g. miR-9-mediated repression of TLX expression in neural stem cells revealed by Western blot ( f ) and RT-PCR ( g ) analyses. A miR-9 mutant with mutations in the seed region was included as a control.

    Journal: Nature structural & molecular biology

    Article Title: A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

    doi: 10.1038/nsmb.1576

    Figure Lengend Snippet: miR-9-directed repression of TLX expression a. miR-9 expression in adult mouse neural stem cells during a differentiation time course. Day 0 (d0) represents the undifferentiated neural stem cell state. U6 was included as a loading control. Relative miR-9 levels, normalized to U6 levels, are indicated under the blots, with miR-9 level in d0 designated as 1. b. Western blot analysis of TLX expression in the same differentiation time course. GAPDH was included as a loading control. c. miR-9-mediated repression of luciferase reporter gene upstream of TLX 3′ UTR. The TLX 3′ UTR reporter gene was co-transfected with increasing amounts of miR-9 RNA duplexes or control siRNA duplexes into HEK 293 cells. d. Similar reporter assays were performed in cells transfected with MDH1-PGK-GFP control vector (1) or miR-9-1 expression vector (2). e. Mutation of the miR-9 target site in TLX 3′ UTR abolished miR-9-mediated repression. Luciferase reporter gene under the control of wild type (WT) or mutant (Mut) TLX 3′ UTR was transfected along with control siRNA (C), a miR-9 mutant with mutations in the seed region complementary to the mutant TLX 3′ UTR, or wild type miR-9 into HEK 293 cells. s.d. is indicated by error bars. f, g. miR-9-mediated repression of TLX expression in neural stem cells revealed by Western blot ( f ) and RT-PCR ( g ) analyses. A miR-9 mutant with mutations in the seed region was included as a control.

    Article Snippet: The mutant miR-9 RNA duplex sense sequence is UGA AAC CAA AUC UAG CUG UAU GA. Western blot of TLX and GAPDH was performed using rabbit anti-TLX antibody (1:1,000) and rabbit anti-GAPDH antibody (Santa Cruz, 1:1,000).

    Techniques: Expressing, Western Blot, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    Overexpression of miR-9 regulates neural stem cell proliferation and differentiation a. Cell proliferation in miR-9-transfected neural stem cells as revealed by BrdU labeling (green). Merged panels show BrdU staining along with phase contrast images. A miR-9 mutant with mutations in the seed region was included as a control, with a total of 200 nM miRNAs in each transfection. b. Quantification of BrdU-positive (BrdU+) cells in miR-9-treated neural stem cells with s.d. indicated by error bars. * p=0.0008 by one-way Anova. c. Top panel is Western blot analysis of TLX expression in miR-9 transfected neural stem cells. GAPDH was included as a loading control. Lower panel is RT-PCR analysis of TLX and p21 in miR-9-transfected neural stem cells. Actin was included as a loading control. d. Overexpression of miR-9 promotes glial differentiation. Control RNA or miR-9-transfected cells were induced into differentiation for 3 days and immunostained with a GFAP-specific antibody (green). Nuclear dapi staining is shown in blue. e. Overexpression of miR-9 promotes neuronal differentiation. Control RNA or miR-9 transfected neural stem cells were induced to differentiate for 3 days and immunostained with a Tuj1-specific antibody (red). Nuclear dapi staining is shown in blue. For both sections d and e , error bars are s.d. of the mean. * p=0.03 ( d ), 0.018 ( e ) by Student's t-test. About 4,000 cells were quantified for panels d and e , respectively.

    Journal: Nature structural & molecular biology

    Article Title: A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

    doi: 10.1038/nsmb.1576

    Figure Lengend Snippet: Overexpression of miR-9 regulates neural stem cell proliferation and differentiation a. Cell proliferation in miR-9-transfected neural stem cells as revealed by BrdU labeling (green). Merged panels show BrdU staining along with phase contrast images. A miR-9 mutant with mutations in the seed region was included as a control, with a total of 200 nM miRNAs in each transfection. b. Quantification of BrdU-positive (BrdU+) cells in miR-9-treated neural stem cells with s.d. indicated by error bars. * p=0.0008 by one-way Anova. c. Top panel is Western blot analysis of TLX expression in miR-9 transfected neural stem cells. GAPDH was included as a loading control. Lower panel is RT-PCR analysis of TLX and p21 in miR-9-transfected neural stem cells. Actin was included as a loading control. d. Overexpression of miR-9 promotes glial differentiation. Control RNA or miR-9-transfected cells were induced into differentiation for 3 days and immunostained with a GFAP-specific antibody (green). Nuclear dapi staining is shown in blue. e. Overexpression of miR-9 promotes neuronal differentiation. Control RNA or miR-9 transfected neural stem cells were induced to differentiate for 3 days and immunostained with a Tuj1-specific antibody (red). Nuclear dapi staining is shown in blue. For both sections d and e , error bars are s.d. of the mean. * p=0.03 ( d ), 0.018 ( e ) by Student's t-test. About 4,000 cells were quantified for panels d and e , respectively.

    Article Snippet: The mutant miR-9 RNA duplex sense sequence is UGA AAC CAA AUC UAG CUG UAU GA. Western blot of TLX and GAPDH was performed using rabbit anti-TLX antibody (1:1,000) and rabbit anti-GAPDH antibody (Santa Cruz, 1:1,000).

    Techniques: Over Expression, Transfection, Labeling, BrdU Staining, Mutagenesis, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining

    miR-9 antisense RNA promotes neural stem cell proliferation a. 2′-O-methyl miR-9 antisense RNA knocks down miR-9 mature form analyzed by Northern blot analysis. 2′-O-methyl antisense GFP RNA was included as a negative control (C) in all sections. U6 was included as a loading control. b. Expression of TLX and p21 in 2′-O-methyl miR-9 antisense RNA-treated neural stem cells analyzed by RT-PCR. GAPDH was included as a loading control. c. Neural stem cells were transfected with control RNA and 2′-O-methyl miR-9 antisense RNA. The transfected cells were labeled by BrdU staining (green). Merged panels show BrdU staining along with phase contrast images. d. Quantification of BrdU+ cells in control (C) and 2′-O-methyl miR-9 antisense RNA-treated neural stem cells. s.d. is represented by error bars. * p=0.03 by Student's t-test.

    Journal: Nature structural & molecular biology

    Article Title: A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

    doi: 10.1038/nsmb.1576

    Figure Lengend Snippet: miR-9 antisense RNA promotes neural stem cell proliferation a. 2′-O-methyl miR-9 antisense RNA knocks down miR-9 mature form analyzed by Northern blot analysis. 2′-O-methyl antisense GFP RNA was included as a negative control (C) in all sections. U6 was included as a loading control. b. Expression of TLX and p21 in 2′-O-methyl miR-9 antisense RNA-treated neural stem cells analyzed by RT-PCR. GAPDH was included as a loading control. c. Neural stem cells were transfected with control RNA and 2′-O-methyl miR-9 antisense RNA. The transfected cells were labeled by BrdU staining (green). Merged panels show BrdU staining along with phase contrast images. d. Quantification of BrdU+ cells in control (C) and 2′-O-methyl miR-9 antisense RNA-treated neural stem cells. s.d. is represented by error bars. * p=0.03 by Student's t-test.

    Article Snippet: The mutant miR-9 RNA duplex sense sequence is UGA AAC CAA AUC UAG CUG UAU GA. Western blot of TLX and GAPDH was performed using rabbit anti-TLX antibody (1:1,000) and rabbit anti-GAPDH antibody (Santa Cruz, 1:1,000).

    Techniques: Northern Blot, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Labeling, BrdU Staining