Journal: Human reproduction (Oxford, England)

Article Title: The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester.

doi: 10.1093/humrep/det258

Figure Lengend Snippet: Figure 2 Relative expression intensity of CXCL6 mRNA. The CXCL6transcriptincreasedinhumanplacentawithpregnancydevelop- ment. CXCL6 transcript levels were examined by RT–PCR in human placenta derived from the first (1, n ¼ 10), second (2, n ¼ 10) or third (3, n ¼ 10) trimesters (T), as indicated, as well as in HTR8/ SVneo cells. GAPDH was used as a control for input. The pictures are representativeexamples fromtenindependent experiment for different tissuesamples.Theresultsinthebar-chart (mean +SEM,n ¼ 10)were calculated from values of CXCL6 mRNA relative to that of GAPDH. *P , 0.05, **P , 0.01 versus 1T.

Article Snippet: Explants were next supplemented with 0.2 ml medium in the absence or presence of different doses (10, 50, 100 or 500 ng/ml) of recombinant human CXCL6 (rhCXCL6; R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Control

Journal: Human reproduction (Oxford, England)

Article Title: The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester.

doi: 10.1093/humrep/det258

Figure Lengend Snippet: Figure 3 Immunohistochemical localization of CXCL6 and its receptors at the maternal–fetal interface. In the first trimester, the protein of CXCL6 was positiveincytotrophoblastsandsyncytiotrophoblastsofvilli (a)andindecidualcells,ininvadingextravilloustrophoblastsofdecidualtissues(b).TheCXCL6 receptors, CXCR1/2 proteins, were stained in villous (c and e) and decidual cells (d and f). Negative control is g. Pictures are representative of five inde- pendent experiments for different tissue samples (villi or deciduas, n ¼ 10).

Article Snippet: Explants were next supplemented with 0.2 ml medium in the absence or presence of different doses (10, 50, 100 or 500 ng/ml) of recombinant human CXCL6 (rhCXCL6; R&D Systems).

Techniques: Immunohistochemical staining, Staining, Negative Control

Journal: Human reproduction (Oxford, England)

Article Title: The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester.

doi: 10.1093/humrep/det258

Figure Lengend Snippet: Figure 6 RhCXCL6 significantly reduced HTR8/SVneo cell migration in a dose-dependent manner. HTR8/SVneo cells were treated with rhCXCL6 at the indicated doses (1, 5, 10, 25, 50, 100 ng/ml) (d–i), with (b) or without CXCL6 blocking antibody (c) and subjected to the migration assay. Controls werenottreatedwithrhCXCL6(0 ng/ml)(a).Resultsareshownasthemeanmigration(expressedasfoldchangefromcontrol)+ SEM,n ¼ 3.**P , 0.01 versus control (0 ng/ml). Original magnification, ×200.

Article Snippet: Explants were next supplemented with 0.2 ml medium in the absence or presence of different doses (10, 50, 100 or 500 ng/ml) of recombinant human CXCL6 (rhCXCL6; R&D Systems).

Techniques: Migration, Blocking Assay, Control

Journal: Human reproduction (Oxford, England)

Article Title: The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester.

doi: 10.1093/humrep/det258

Figure Lengend Snippet: Figure 8 RhCXCL6 significantly reduced the invasive ability of primary trophoblasts. Primary trophoblasts were treated without (control) (a) or with rhCXCL6 (100 ng/ml) (b), with (c) or without (d) CXCL6 blocking antibody, and subjected to the invasion assay. Results are shown as the mean invasion (expressed as fold change from control) +SEM. *P , 0.05 versus control (0 ng/ml). Original magnification, ×200.

Article Snippet: Explants were next supplemented with 0.2 ml medium in the absence or presence of different doses (10, 50, 100 or 500 ng/ml) of recombinant human CXCL6 (rhCXCL6; R&D Systems).

Techniques: Control, Blocking Assay, Invasion Assay

Journal: Human reproduction (Oxford, England)

Article Title: The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester.

doi: 10.1093/humrep/det258

Figure Lengend Snippet: Figure 9 Effect of CXCL6 on secreted MMP-2 and MMP-9 levels. ProMMP-2 levels are suppressed in supernatants of rhCXCL6-treated HTR8/SVneo cells. HTR8/SVneo cells were treated with rhCXCL6 (1, 5, 10, 25, 50, 100 ng/ml); 24 h later supernatants were collected and analysed for the levels of ProMMP-2(72 kDa) andProMMP-9(92 kDa)and comparedwith non-treatedcontrols(0 ng/ml).Resultsareshownasthemean(relativeexpressionlevel of proMMP-2/-9) + sem fold change compared with control, n ¼ 3. *P , 0.05, **P , 0.01 compared with control (0 ng/ml). Active MMP-9 and MMP-2 enzymes were not detected.

Article Snippet: Explants were next supplemented with 0.2 ml medium in the absence or presence of different doses (10, 50, 100 or 500 ng/ml) of recombinant human CXCL6 (rhCXCL6; R&D Systems).

Techniques: Control

Journal: Human reproduction (Oxford, England)

Article Title: The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester.

doi: 10.1093/humrep/det258

Figure Lengend Snippet: Figure 10 Effect of CXCL6 on secreted MMP-2 and MMP-9 levels. ProMMP-2 levels are significantly suppressed in supernatants of rhCXCL6-treated primary trophoblasts. Primary trophoblasts were treated with rhCXCL6 (100 ng/ml); 24 h later supernatants were col- lected and analysed for the levels of Pro-MMP-2 (72 kDa) and Pro-MMP-9 (92 kDa), and compared with non-treated controls (0 ng/ml).Resultsareshownasthemean + SEMfoldchangecompared with control. *P , 0.05 compared with control (0 ng/ml). Active MMP-9 and MMP-2 enzymes were not detected. Experiment is repre- sentative of three independent experiments.

Article Snippet: Explants were next supplemented with 0.2 ml medium in the absence or presence of different doses (10, 50, 100 or 500 ng/ml) of recombinant human CXCL6 (rhCXCL6; R&D Systems).

Techniques: Control

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

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

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 1. RASEF expression in tumor tissues and cell lines. A, expression of RASEF in 12 clinical lung cancers (T; 4 clinical lung ADC, 4 clinical lung SCC, and 4 clinical SCLC) and corresponding normal lung tissues (N) detected by semiquantitative RT-PCR analysis. B, expression of RASEF in 22 lung cancer cell lines and a bronchial epithelial cell line BEAS-2B detected by semiquantitative RT-PCR analysis. ASC indicates lung adenosquamous cell carcinoma; LCC, large cell carcinoma. C, Western blot analysis of RASEF protein using anti-RASEF antibody. IB, immunoblotting. D, expression and subcellular localization of endogenous RASEF protein in RASEF-positive and RASEF-negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in A549 and NCI-H2170 cells, whereas no staining was observed in DMS114 and bronchial epithelia–derived BEAS-2B cell lines.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 4. Enhanced phosphorylation of ERK1/2 by RASEF in lung cancer cells. A, expression of MAPK signal molecules and their phosphorylation levels in DMS114 cells transfected with RASEF expression vector or mock plasmid. B, expression of MAPK signal molecules and their phosphorylation levels in NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC). C and D, expression levels of downstream target genes of MAPK cascade were regulated by RASEF expression in lung cancer cells. Total RNA from BEAS-2B and DMS114 cells transfected with RASEF expression vector or mock plasmid (C) and A549 and NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC; D) were subjected to reverse-transcription reaction, followed by PCR reaction to evaluate the expression levels of CCND1, CCNB1, and CDKN1A transcription. Western blotting with antiphosphorylated ERK1/2 antibody was conducted to confirm the change of ERK1/2 phosphorylation according to RASEF expression.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 5. Identification of ERK1/2-interacting sites on RASEF. A, interaction of endogenous RASEF with endogenous ERK1/2. The immunoprecipitates obtained using anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. B, schematic representation of various partial constructs of RASEF expression vector. C and D, determination of the ERK1/2-interacting regions on RASEF by immunoprecipitation experiments using DMS114 cells transfected with vectors expressing partial RASEF protein. COOH-terminal part of RASEF (codons 520–575) was likely to be ERK1/2-interacting region.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection

Journal: Stem cells (Dayton, Ohio)

Article Title: Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors.

doi: 10.1002/stem.1622

Figure Lengend Snippet: Figure 1. Differentiation of human pluripotent stem cells to neuroepithelium under defined conditions. (A): Experimental timeline. (B): Reverse transcriptase polymerase chain reaction analysis of pluripotency, mesoderm, endoderm, and neuroectoderm gene expres- sion in differentiating H9 human embryonic stem cells (hESCs). “SB” indicates addition of SB431542 and “N” indicates addition of nog- gin. (C): Flow cytometry analysis of Pax6. Data are presented as mean 6 SD calculated from at least two biological replicates. Differentiation was conducted on Matrigel-coated substrates unless otherwise specified. (D): Images of neural rosette formation on day 6 of H9 hESC differentiation. The inset shows the magnified rosette structure. All images are of cells differentiated on Matrigel- coated substrates except for the one labeled “E6 (VTN-NC),” which indicates cells differentiated in E6 medium on substrates coated with recombinant vitronectin peptide. Scale bars in Pax6/N-cadherin-stained images are 250 mm; scale bars in Otx2/Sox2-stained images are 50 mm. (E): Representative flow cytometry histograms of N-cadherin, Otx2, and Sox2 expression at day 6 of differentiating H9 hESCs in E6 medium. Data are representative of two biological replicates and mean 6 SD are listed in Results. Gray histogram, IgG control; red histogram, label of interest. (F): Progression of Sox1 expression in E6/FI conditions with and without retinoic acid. Scale bars 5 100 mm.

Article Snippet: The following morning, cells were changed to E6 medium, E6 containing 10 mM SB431542 (Cellagentech, San Diego, CA (www.cellagen tech.com)), or E6 containing 10 mM SB431542 and 200 ng/ml recombinant human noggin (R&D Systems) to initiate differentiation.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Labeling, Recombinant, Staining, Cytometry, Expressing, Control

Journal: International journal of oncology

Article Title: USP22 promotes the G1/S phase transition by upregulating FoxM1 expression via β-catenin nuclear localization and is associated with poor prognosis in stage II pancreatic ductal adenocarcinoma.

doi: 10.3892/ijo.2014.2531

Figure Lengend Snippet: Figure 6. USP22 expression affects FoxM1 expression and Wnt/β-catenin pathway activation. (A) Triple IF staining for FoxM1 (red), β-catenin (green) and nuclei (DAPI, blue) was performed on PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM). (B) Triple IF staining for FoxM1 (red), β-catenin (green) and nuclei (DAPI, blue) was performed on CFPAC-1 cells that were transfected by vector USP22-1 (2 µg) or USP22-2 (10 µg). (C and D) RT-PCR and western blot analysis of FoxM1 and β-catenin in PANC-1 cells that were transfected by control- siRNA, USP22‑siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM) (left panel) and CFPAC-1 cells that were transfected by vector, USP22-1 (2 µg) or USP22-2 (10 µg) (right panel). (E) Cellular levels of Axin2, c-Myc and LEF-1 in PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22‑siRNA-2 (150 nM) (left panel) and CFPAC-1 cells that were transfected by vector, USP22-1 (2 µg) or USP22-2 (10 µg) (right panel). (F) Activities of TOP-Flash and FOP-Flash in PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM) (left panel) and CFPAC-1 cells that were transfected by vector, USP22-1 (2 µg) or USP22-2 (10 µg) (right panel). (G) Cytoplasmic and nuclear levels of FoxM1 and β-catenin in PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM). (H) Cytoplasmic and nuclear levels of FoxM1 and β-catenin in CFPAC-1 cells that were transfected by vector USP22-1 (2 µg) or USP22-2 (10 µg).

Article Snippet: Membranes were blocked in a buffer (TBS: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5% bovine serum albumin and 0.1% Tween-20, followed by incubation with the primary antibodys USP22 (ab4812, 1:2,000, Abcam), FoxM1 (SC-502, 1:200, Santa Cruz Biotechnology), cyclin D1 (60186-1-lg, 1:100), p21 (10355-1-AP, 1:100), p27 (10567-1-AP, 1:100), cdk4 (11026-1-AP, 1:500), cdk6 (14052-1-AP, 1:500, all from Proteintech, Chicago, IL, USA), β-catenin (SC-7963, 1:500, Santa Cruz Biotechnology), Axin2 (EPR2005-2, 1:2,000), LEF (EP2030Y, 1:2,000, both from Abcam), c-Myc (10057-1-AP, 1:200) and LMNB1 (12987-1-AP, 1:1,000) or β-actin (20536-1-AP, 1:2,000, all from Proteintech) diluted in the same buffer.

Techniques: Expressing, Activation Assay, Staining, Transfection, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 1 Multiple sequence alignment of TMEM106B protein. Multiple amino acid sequence alignment was performed by importing the corresponding amino acid sequences into CLC Free Workbench (CLC Bio/Qiagen, Aarhus, Denmark). (a) Multiple amino acid sequence alignment of TMEM106B orthologs derived from Homo sapiens, Pan troglodytes, Canis lupus familiaris, Bos Taurus, Mus musclus, Rattus norvegicus, Gallus gallus, Danio rerio, and Xenopus laevis. (b) Multiple amino acid sequence alignment of the human TMEM106A, TMEM106B, and TMEM106C proteins.

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Sequencing, Derivative Assay

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 2 Universal expression of TMEM106B mRNAs in human neural cells. mRNA expression was studied by reverse transcriptase (RT)-polymerase chain reaction (PCR) in human tissues and cultured cells. (a) TMEM106A, (b) TMEM106B, (c) TMEM106C, (d) progranulin (PGRN), and (e) G3PDH, a housekeeping gene for a positive control. The lanes indicate (1) the frontal cortex of the human cerebrum (CBR) with inclusion of the RT step, (2) CBR without inclusion of the RT step, (3) astrocytes (AS), (4) neuronal progenitor (NP) cells, (5) NTera2 teratocarcinoma-derived neurons, (6) SK-N-SH neuroblastoma, (7) IMR-32 neuroblastoma, (8) U-373MG glioblastoma, (9) T98G glioblastoma, and (10) HMO6 microglia. TMEM106A, TMEM106B, TMEM106C, and PGRN were amplified for 35 cycles, while G3PDH was amplified for 28 cycles.

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Cell Culture, Positive Control, Derivative Assay, Amplification

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 3 Reduced expression of TMEM106B mRNA in Alzheimer’s disease brains. TMEM106B and progranulin (PGRN) mRNA expression levels were studied by quantitative reverse transcriptase-polymerase chain reaction (qPCR) in human brain tissues derived from a reference of the human frontal cortex (REF), four non-neurological control cases (NC), six amyotrophic lateral sclerosis (ALS) cases, four Parkinson’s disease (PD) cases, and seven Alzheimer’s disease (AD) cases. The expression levels were standardized against those of G3PDH. (a) TMEM106B mRNA expression. (b) PGRN mRNA expression. (c) Difference in TMEM106B levels between AD and non-AD cases. *P = 0.0035 by Student’s t test. (d) Difference in PGRN levels between AD and non-AD cases. **P = 0.0027 by Student’s t test. (e) Pearson’s correlation between TMEM106B and PGRN mRNA levels. Pearson’s correlation coefficient indicates −0.555 (P = 0.0090).

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Derivative Assay, Control

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 4 Positive correlation between TMEM106B and neurofilament, heavy polypeptide mRNA levels. Neurofilament, heavy polypeptide (NFH), glial fibrillary acidic protein (GFAP), and RNA-binding protein, fox-1 homolog (Caenorhabditis elegans)-3 (RBFOX3, NEUN) mRNA expression levels were studied by quantitative reverse transcriptase-polymerase chain reaction (qPCR) in human brain tissues derived from a reference of the human frontal cortex (REF), four non-neurological causes (NC) cases, six amyotrophic lateral sclerosis (ALS) cases, four Parkinson’s disease PD cases, and seven AD cases. The expression levels were standardized against those of G3PDH. (a) NFH expression. (b) GFAP expression. (c) NEUN expression. (d) Pearson’s correlation between TMEM106B and NFH mRNA levels. Pearson’s correlation coefficient indicates 0.496 (P = 0.0221).

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: RNA Binding Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction, Derivative Assay

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 5 Characterization of anti-TMEM106B antibody. The full-length open reading frame (ORF) cloned in the vector that expresses a fusion protein with an N-terminal Xpress tag was transiently expressed in HeLa cells. Total protein extract was processed for western blot. Lanes represent the protein of (1) untransfected cells and the cells expressing (2) TMEM106A, (3) TMEM106B, or (4) TMEM106C, and the protein of (5) human brain #1, (6) human brain #2, or (7) IMR-32 neuroblastoma cells. Immunoblots of (a, d) TMEM106B (the A303-439A antibody), (b) Xpress, and (c, e) HSP60, an internal control for protein loading.

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Clone Assay, Plasmid Preparation, Western Blot, Expressing, Control

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 6 Reduced expression of TMEM106B protein in Alzheimer’s disease brains. Protein expression levels were studied by western blot in human brain tissues derived from four non-neurological causes (NC) cases, six amyotrophic lateral sclerosis (ALS) cases, four Parkinson’s disease (PD) cases, and seven Alzheimer’s disease (AD) cases. The expression levels were standardized against those of HSP60. (A) TMEM106B expression: (a) TMEM106B and (b) HSP60. (B) Progranulin (PGRN) expression: (a) PGRN and (b) HSP60. (C) Difference in TMEM106B levels between AD and non-AD cases. *P = 0.0000004 by Student’s t test. (D) Difference in PGRN levels between AD and non-AD cases. ns, non-significant (P = 0.5304 by Student’s t test). (E) Pearson’s correlation between TMEM106B and PGRN protein levels. Pearson’s correlation coefficient indicates −0.242 (P = 0.2912).

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Expressing, Western Blot, Derivative Assay

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 7 TMEM106B immunoreactivity in non-Alzheimer’s disease brains. Expression of TMEM106 immunoreactivity was studied in 13 non-Alzheimer’s disease brains presented in Table 1 by immunohistochemistry using the A303-439A antibody. (a) Non-neurological causes (NC), the frontal cortex, cytoplasmic staining of cortical neurons; (b) amyotrophic lateral sclerosis (ALS), the frontal cortex, cytoplasmic staining of cortical neurons; (c) NC, the hippocampal CA1 region, cytoplasmic staining of pyramidal neurons; (d) ALS, the hippocampal CA1 region, cytoplasmic staining of pyramidal neurons; (e) NC, the hippocampal CA1 region, intense staining of small nodular structures accumulated in the perinuclear region of pyramidal neurons; (f) NC, the frontal white matter, cytoplasmic staining of oligodendrocytes, reactive astrocytes, and microglia.

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Alzheimer's research & therapy

Article Title: TMEM106B expression is reduced in Alzheimer's disease brains.

doi: 10.1186/alzrt247

Figure Lengend Snippet: Figure 8 TMEM106B and PGRN immunoreactivities in Alzheimer’s disease brains. Expression of TMEM106 and progranulin (PGRN) immunoreactivities was studied in six Alzheimer’s disease brains presented in Table 1 by immunohistochemistry using the A303-439A antibody. (a) TMEM106B, the frontal cortex, moderate neuronal cytoplasmic staining and faint senile plaque staining; (b) PGRN, same region as (a), moderate senile plaque staining and diffuse neuropil staining; (c) TMEM106B, the hippocampal CA1 region, intense neuronal and astroglial cytoplasmic staining; (d) PGRN, same region as (c), intense perivascular neuropil staining; (e) TMEM106B, the hippocampal CA1 region, no staining of senile plaques and neurofibrillary tangles; (f) PGRN, same region as (e), moderate staining of numerous senile plaques and neurofibrillary tangles.

Article Snippet: After gel electrophoresis, the protein was transferred onto nitrocellulose membranes, followed by incubation at room temperature overnight with the anti-TMEM106B antibody A303-439A, rabbit polyclonal anti-TMEM106B antibody raised against a peptide spanning amino acid residues 101 to 200 of the human TMEM106B protein (bs-11694R; Bioss, Boston, MA, USA), rabbit polyclonal anti-TMEM106B antibody raised against the human TMEM106B-GST fusion protein (20995-1-AP; Proteintech, Chicago, IL, USA), or mouse monoclonal anti-Xpress antibody (Invitrogen).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Journal of pharmacological sciences

Article Title: Inhibition of smooth muscle cell proliferation by ezetimibe via the cyclin D1-MAPK pathway.

doi: 10.1254/jphs.13239fp

Figure Lengend Snippet: Fig. 2. Ezetimibe inhibits cell cycle progression by regulating activity and expression of cell cycle–related proteins. A) Ezetimibe downregulates the promoter activity of cyclin D1 in VSMCs induced by Chol:MbCD. VSMCs were treated as indicated for 72 h and then the promoter activity of cyclin D1 was detected by luciferase assays. *P < 0.01 vs. Chol:MbCD. B) Ezetimibe downregulates the protein expression of cyclin D1, CDK4, p-RB, and E2F in VSMCs stimulated by Chol:MbCD. Protein expression was detected by western blot analysis (30 mg protein/lane). a-Tublin was used as a loading control. *P < 0.05 vs. Control; #P < 0.05 vs. Chol:MbCD; **P < 0.05 vs. Chol:MbCD. C) Ezetimibe downregulates the mRNA level of cyclin D1 in VSMCs induced by Chol:MbCD. VSMCs were treated as indicated for 72 h and then mRNA level was assayed by RT-PCR. GAPDH was used as the control. *P < 0.01 vs. Chol:MbCD. The data represent 3 independent experiments.

Article Snippet: The primary antibodies for MEK1, phospho-MEK1 (p-MEK1), CDK4, cyclin D1, E2F, and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Expressing, Luciferase, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of pharmacological sciences

Article Title: Inhibition of smooth muscle cell proliferation by ezetimibe via the cyclin D1-MAPK pathway.

doi: 10.1254/jphs.13239fp

Figure Lengend Snippet: Fig. 3. Ezetimibe regulates cyclin D1-MAPK signaling in VSMCs treated by Chol: MbCD. A) The localization of p-ERK1/2 (red) was determined by immunofluorescence and nuclear staining by DAPI (blue). B) VSMCs were incubated with or without 3 mM ezetimibe for 72 h in the presence of 10 mg/mL Chol:MbCD followed by treatment with the ERK1/2 inhibitor PD98059 (50 mM) for 1 h. The expression of cyclin D1, p-ERK1/2, total ERK1/2, p-MEK1, total MEK1, and a-tublin were measured by western blot analysis. **P < 0.01 vs. control group (without treatment of Chol:MbCD, ezetimibe, and PD98059); *P < 0.05 vs. Chol:MbCD treatmen alone group; #P < 0.05 vs. PD98059 treatment alone group. The data represent 3 independent experiments.

Article Snippet: The primary antibodies for MEK1, phospho-MEK1 (p-MEK1), CDK4, cyclin D1, E2F, and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunofluorescence, Staining, Incubation, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

doi: 10.1371/journal.pone.0041076

Figure Lengend Snippet: Hearts were perfused with an inhibitor of p38 MAPK (SB203580, 1 µM) 5 min prior to and during perfusion with 100 nM PPT. A. 1 min PPT. B. 2.5 min PPT. C. 5 min PPT. Inhibition of p38 MAPK blocked the effects of PPT on actomyosin MgATPase activity at all time points. D. Treatment with SB203580 had no effect on myofilament activation at all timepoints. N = 5 for all groups.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder (total p38 MAPK) and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Inhibition, Activity Assay, Activation Assay

Journal: PLoS ONE

Article Title: Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

doi: 10.1371/journal.pone.0041076

Figure Lengend Snippet: Antagonism of the effects of ERα activation on cardiac myofilament function with a p38 MAPK inhibitor.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder (total p38 MAPK) and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

doi: 10.1371/journal.pone.0041076

Figure Lengend Snippet: Hearts were perfused with the p38 MAPK inhibitor SB203580 (1 µM) for 5 min before and during PPT (100 nM) treatment. Hearts were exposed to PPT for up to 5 min and cardiac myofilament isolated. A. Isolated cardiac myofilaments were separated by SDS-PAGE on a 12% gel. Gels were stained with Pro-Q Diamond Phosphoprotein gel stain to assess global phosphorylation. Equal protein loading was confirmed by Coomassie staining. The actin band from Coomassie stained gels is shown as representative of protein loading. B. Analysis of gels for global phosphorylation of myosin-binding protein C (MyBP-C), troponin T (cTnT), tropomyosin (Tm), and troponin I (cTnI) revealed no significant changes in phosphorylation status of any myofilament protein. N = 5 for all groups.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder (total p38 MAPK) and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Isolation, SDS Page, Staining, Binding Assay

Journal: PLoS ONE

Article Title: Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

doi: 10.1371/journal.pone.0041076

Figure Lengend Snippet: Hearts were perfused with the p38 MAPK inhibitor SB203580 (1 µM) for 5 min, followed by treatment with 100 nM PPT for 1, 2.5 or 5 minutes in the presence of SB203580. Isolated cardiac myofilaments were separated by SDS-PAGE on a 10% gel. A. Composite blot of cTnI phosphorylation (serines 22/23) as measured by immunoblot analysis and normalized to total actin. B. cTnI phosphorylation at serines 22/23 was significantly less than control levels at all treatment times. N = 4.*P<0.05 vs Control.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder (total p38 MAPK) and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Isolation, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

doi: 10.1371/journal.pone.0041076

Figure Lengend Snippet: Hearts were perfused with 100 nM PPT for 1, 2.5 or 5 minutes. A. p38 activation was assessed with immunoblot analysis of myocardium homogenates with an antibody against phosphorylated p38, and normalized to total p38. B. p38 activity was significantly increased following 2.5 minutes of ERα activation (N = 4, p<0.05) and remained elevated at 5 min. N = 4. SB203580 (1 µM) inhibited the PPT-dependent increase in phosphorylated p38 at all time points (N = 4 for all time points). *P<0.05 vs Control.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder (total p38 MAPK) and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

doi: 10.1371/journal.pone.0041076

Figure Lengend Snippet: Hearts were perfused on a Langendorff apparatus to establish baseline pressures. The final 5 min (-5 to 0 min) before PPT treatment were taken as the baseline. Some hearts were treated with 1 µM SB203580 to inhibit p38 MAPK. PPT treatment had no significant effect on A. left ventricular end systolic or B. diastolic pressure. Neither C. rate of pressure development nor D. rate of relaxation were altered by PPT. p38 MAPK inhibition with SB203580 did not differ from controls in any parameter. (N = 6 for all time points). *P<0.05 vs Control.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder (total p38 MAPK) and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Inhibition

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of N-cadherin proteins in myocardial hypertrophy in rats

doi: 10.3892/etm.2013.1431

Figure Lengend Snippet: Oligonucleotide primers of N-cadherin and β-actin.

Article Snippet: Primary antibodies of N-cadherin (1:100; Wuhan Boster Biological Technology Ltd., Wuhan, China) were incubated overnight at 4°C and subsequently incubated at 37°C for 30 min in biotin-labeled IgG and streptavidin-biotin complex liquid.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of N-cadherin proteins in myocardial hypertrophy in rats

doi: 10.3892/etm.2013.1431

Figure Lengend Snippet: N-cadherin expression in the myocardial tissue by immunohistochemical method. (A) Control group. (B) Experimental group. Magnification, ×400.

Article Snippet: Primary antibodies of N-cadherin (1:100; Wuhan Boster Biological Technology Ltd., Wuhan, China) were incubated overnight at 4°C and subsequently incubated at 37°C for 30 min in biotin-labeled IgG and streptavidin-biotin complex liquid.

Techniques: Expressing, Immunohistochemical staining, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of N-cadherin proteins in myocardial hypertrophy in rats

doi: 10.3892/etm.2013.1431

Figure Lengend Snippet: Comparison of the average area and brightness of N-cadherin expression in the myocardium (mean ± standard deviation).

Article Snippet: Primary antibodies of N-cadherin (1:100; Wuhan Boster Biological Technology Ltd., Wuhan, China) were incubated overnight at 4°C and subsequently incubated at 37°C for 30 min in biotin-labeled IgG and streptavidin-biotin complex liquid.

Techniques: Comparison, Expressing, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of N-cadherin proteins in myocardial hypertrophy in rats

doi: 10.3892/etm.2013.1431

Figure Lengend Snippet: N-cadherin expression in the myocardial tissue by immunofluorescence method. (A) Control group. (B) Experimental group. Magnification, ×400.

Article Snippet: Primary antibodies of N-cadherin (1:100; Wuhan Boster Biological Technology Ltd., Wuhan, China) were incubated overnight at 4°C and subsequently incubated at 37°C for 30 min in biotin-labeled IgG and streptavidin-biotin complex liquid.

Techniques: Expressing, Immunofluorescence, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of N-cadherin proteins in myocardial hypertrophy in rats

doi: 10.3892/etm.2013.1431

Figure Lengend Snippet: Reverse transcription polymerase chain reaction; products of N-cadherin. M, Marker; 1–2, experimental group; 3–4, control group.

Article Snippet: Primary antibodies of N-cadherin (1:100; Wuhan Boster Biological Technology Ltd., Wuhan, China) were incubated overnight at 4°C and subsequently incubated at 37°C for 30 min in biotin-labeled IgG and streptavidin-biotin complex liquid.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Marker, Control

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Expression of proHB-EGF under the control of the villin promoter. (A) Villin vector. The vector consists of the 12.4 kb villin promoter sequence and the SV40 polyA signals and intron. The Mlu I and SacII sites where the HB-EGF sequence has been inserted are shown. The two PmeI cut sites for release of the entire construct are also labeled. The asterisk represents the initiation site for transcription of the villin sequences. (B) Villin-proHB-EGF construct. Human precursor HB-EGF cDNA (625 bp) was inserted into the Mlu I and SacII sites at the 3′ end of the villin promoter. (C) PCR of potential HB-EGF TG founder mice. Eight potential founder mice derived from Vill-HB-EGF-injected oocytes were screened for genomic integration of the HB-EGF transgene by PCR of tail DNA using villin promoter and HB-EGF sequence specific primers (top panel). Mouse specific β-actin primers were used as an internal control for amplification (bottom panel). Two HB-EGF TG mice were identified (lanes 2 and 4). c, control DNA (Villin-proHB-EGF). (D) Southern blot analysis of potential HB-EGF TG founder mice. DNA was isolated from the tails of the same eight potential founder mice and subjected to Southern blot analysis using HB-EGF specific probes. The positive bands in lanes 2 and 4 correlated with the PCR results, confirming the identification of two HB-EGF TG mice. C, FVB control DNA.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Control, Plasmid Preparation, Sequencing, Construct, Labeling, Derivative Assay, Injection, Amplification, Southern Blot, Isolation

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Overexpression of HB-EGF in TG mouse intestine. (A) human HB-EGF mRNA expression as determined by RT-PCR. Tissues from nine different organs were harvested from 1-month-old TG and WT mice and RNA was isolated. RT-PCR using villin- and HB-EGF-specific primers was performed (upper panel). RT-PCR for β-actin was performed in parallel as an internal control of the amplification signal (lower panel). Lanes 12–15, (−)RT controls. (B) human HB-EGF mRNA expression determined by real time RT-PCR. HB-EGF expression in TG mice was determined using real time RT-PCR with specific primers to hHB-EGF that do not amplify murine HB-EGF. Note that the Y-axis is shown in a log scale. (C) HB-EGF protein production as determined by immunoprecipitation and Western blotting. Transgene protein product from jejunal lysates (2 mg) was immunoprecipitated with a goat anti-HB-EGF polyclonal antibody (20 μg) and subsequently detected by Western blotting. WT jejunum served as a negative control (Lane 1). Recombinant mature HB-EGF protein (19 kD) spiked into WT jejunal lysates was immunoprecipitated with goat IgG to serve as an additional negative control (Lane 8) or with goat anti-HB-EGF antibodies to serve as a positive control (Lane 9). RT-PCR demonstrating the corresponding human HB-EGF transgene expression for the lanes in Panel C. WT, wild type; low, 1–low 4, intestine from four individual low expression TG mice; high 1 and high 2, intestine from two individual high expression TG mice. (D) Immunofluorescent staining of transgene human HB-EGF protein in the jejunum using anti-human-HB-EGF antibodies. Positively stained cells were stained red (Cy3). Tissues were counterstained with DAPI (blue). White arrowheads indicate perinuclear HB-EGF staining. Upper panels, magnification 200 ×; lower panels, magnification 400 ×. (E) Immunofluorescent staining of transgene human HB-EGF protein in the colon. WT, wild type; low, low expression HB-EGF TG mice; high, high expression HB-EGF TG mice. Scale bars represent 50 μm.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Amplification, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Negative Control, Recombinant, Positive Control, Staining

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Body weight determinations of HB-EGF TG and WT mice. Body weights of male and female TG mice and WT littermates were determined weekly from 4–12 weeks of life. N = 35 animals per group.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques:

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Intestinal histology and morphometric analysis of HB-EGF TG and WT mice. (A) Intestinal histology. Shown are representative H&E-stained sections of duodenum from WT, high expression TG mice, and low expression TG mice at 1 month and 5 months of age. The scale bar represents 100 μm. (B) Intestinal morphometric analysis. Villous length, villous width, crypt depth, and muscle thickness in H&E-stained sections of duodenum, jejunum, and ileum from WT, high expression TG mice, and low expression TG mice were quantified using ImageJ 1.39U software. (C) Enterocyte immunostaining. Shown are jejunal sections from WT, high expression TG, and low expression TG mice immunostained with rabbit anti-E-cadherin and secondary goat anti-rabbit IgG conjugated with Alexa 560 (red). The nulei were conterstained with DAPI (blue). The scale bar represents 50 μm. (D) Enterocyte cell density. Columnar enterocyte cell densities were quantified by counting the number of cells in the distal 200 μm from the distal tips of the villi. Each graph bar represents quantification of 15 villous tips. All values shown represent mean ± SD. Statistical significance was determined by one-way ANOVA (repeated measures). * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; WT, wild type; low, low expression HB-EGF TG mice; high, high expression HB-EGF TG mice.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Expressing, Software, Immunostaining

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Crypt cell proliferation in HB-EGF TG and WT mice. (A) BrdU immunostaining. Shown are representative jejunual sections from 1-month-old WT, high expression TG, and low expression TG mice subjected to BrdU immunostaining. Proliferating cells are stained red-brown. (B) Quantification of crypt cell proliferation. Proliferation indices were calculated as the percent (%) of BrdU positive nuclei/total crypt nuclei. Nuclei in at least 15 crypts from the duodenum, jejunum, and ileum were measured to determine proliferation indices. (C) SC IHC. SCs in jejunal crypts of 1-month-old WT mice are shown. SCs were labeled with rat anti-prominin-1 monoclonal antibody (red staining). (D) Quantification of SCs. Prominin-1 positive cells beneath cell +4 level in 15 crypts were counted. (E) Proliferative SCs in crypts (arrows). BrdU positive cells beneath cell +4 position were scored. The scale bars in A, C, and E represent 50 μm. Values shown are mean ± SD. Statistical significance was determined by one-way ANOVA (repeated measures). * p < 0.05; ** p < 0.01; **** p < 0.001; WT, wild type; low, low expression HB-EGF TG mice; high, high expression HB-EGF TG mice.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunostaining, Expressing, Staining, Labeling

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: IEC apoptosis in HB-EGF TG and WT mice. (A) Apoptosis in ileal villi in high expression TG and WT mice at 1 and 5 months of age demonstrated by TUNEL staining (red) and anti-cleaved caspase 3 immunostaining (green), with DAPI nuclear counterstaining (blue). White arrows indicate examples of positively staining cells. The scale bar represents 50 μm. (B) Quantification of apoptotic activity in WT, low expression TG, and high expression TG villi at 1 and 5 months of age. Five to ten high power fields at 200 × magnification with the highest numbers of apoptotic cells by TUNEL staining were photographed at 200 × magnification using a Zeiss microscope and TUNEL positive cells/villus were counted. Values represent mean ± SD. Statistical significance was determined by one-way ANOVA (repeated measures). * p < 0.05; ** p < 0.01; *** p < 0.005; WT, wild type; low, low expression HB-EGF TG mice; high, high expression HB-EGF TG mice.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, TUNEL Assay, Staining, Immunostaining, Activity Assay, Microscopy

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Intestinal goblet cell, neuroendocrine cell, and Paneth cell quantification in HB-EGF TG and WT mice. (A) Goblet cell quantification. Tissue sections were stained with PAS to stain the cytoplasm of goblet cells pink. Quantification of goblet cells in the duodenum, jejunum, and ileum of WT, low expression TG mice, and high expression TG mice was performed by counting the numbers of PAS-positive goblet cells/villous. (B) Neuroedocrine cell quantification. Neuroendocrine cells were immunostained with rabbit anti-chromogranin A antibody, with nuclear counterstaining with DAPI. Quantification of neuroendocrine cells in the jejunum and ileum of WT, low expression TG mice, and high expression TG mice was performed by counting the numbers of positively stained cells/villous. (C) Paneth cell quantification. Paneth cells were identified as granule-containing cells at the base of the crypts of H&E-stained sections, with quantification performed by counting the granule-containing cells/crypt in the jejunum of WT, high expression TG, and low expression TG mice. All values shown represent mean ± SD. Statistical analyses were performed by one-way ANOVA (repeated measures). * p < 0.05; *** p < 0.005; WT, wild type; low, low expression HB-EGFTG mice; high, high expression HB-EGFTG mice.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Expressing

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Analysis of gut-associated lymphoid tissue (GALT) and dendritic cells in HB-EGF TG and WT mice. Lymphocytes in HB-EGF TG and WT mice. Lymphocytes from Peyer’s patches were fluorescently labeled using antibodies specific for CD3, CD4, CD8, and immunoglobulin. GALT was analysed by flow cytometry. The intestinal dendritic cells were visualized by anti-dendritic cell IHC and quantified by counting the numbers of positively stained cells/400 × high power field. Statistical analyses were performed by one-way ANOVA (repeated measures). Values shown are mean ± SD. WT, wild type; high, high expression HB-EGF TG mice.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Labeling, Flow Cytometry, Staining, Expressing

Journal: Growth factors (Chur, Switzerland)

Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

doi: 10.3109/08977190903407365

Figure Lengend Snippet: Overexpression of human HB-EGF in mice subjected to HS/R. HB-EGF TG and WT mice were subjected to hemorrhagic shock (30–35 mmHg) for 90 min, followed by resuscitation for 3 h. Intestinal permeability was determined using the everted gut sac method. *p < 0.05. Values shown are mean ± SD. WT, wild type; high, high expression HB-EGF TG mice.

Article Snippet: Specific bands were visualized using the enhanced chemifluorescence (ECF) western blotting reagent pack (Amersham, Pittsburgh, PA, USA) as previously described ( Chen et al. 2007 ). proHB-EGF protein was detected by probing blots with a goat anti-HB-EGF antibody (0.2 μg/ml; R&D Systems, Minneapolis, MN, USA) or a rabbit anti-HB-EGF antibody (1 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Over Expression, Permeability, Expressing