Journal: Arthritis Research & Therapy

Article Title: Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells

doi: 10.1186/s13075-017-1233-0

Figure Lengend Snippet: Expression of α-Klotho, vascular endothelial growth factor ( VEGF ) 165 b, VEGF receptor-2 ( VEGFR-2 ) and transient receptor potential canonical-1 ( TRPC-1 ) in dermal microvascular endothelial cells from healthy controls ( H-MVECs ) and patients with systemic sclerosis (SSc) ( SSc-MVECs ). a , b Representative microphotographs of immunocytochemical staining for α-Klotho in H-MVECs ( a ) and SSc-MVECs ( b ) detected with biotinylated secondary antibodies and streptavidin peroxidase. c - f Protein lysates from H-MVECs and SSc-MVECs were subjected to western blotting analysis using anti-α-Klotho ( c ), anti-VEGF 165 b ( d ), anti-VEGFR-2 ( e ) and anti-TRPC-1 ( f ) antibodies. Representative immunoblots are shown. The densitometric analysis of the bands normalized to α-tubulin is reported in the histograms. Data are mean ± SD of optical density ( OD ) in arbitrary units. Student’s t test was used for statistical analysis; p values are indicated in each panel. Results are representative of three independent experiments performed with each one of the five H-MVEC and five SSc-MVEC lines

Article Snippet: The day after, tissue sections were washed three times in PBS and incubated with biotinylated secondary antibodies (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes at room temperature.

Techniques: Expressing, Staining, Western Blot

Journal: Nature cell biology

Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

doi: 10.1038/ncb3595

Figure Lengend Snippet: PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense biotinylated probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.

Article Snippet: Sections were incubated overnight in a humid chamber at 4°C with antibody against Ki-67 purchased from Cell signaling technology company (Clone [D2H10], Cat. #9027, 1:1600 dilution) followed by biotinylated secondary antibody (Vector laboratories) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (Dako), and sections were counterstained with hematoxylin before mounting.

Techniques: Fractionation, Reverse Transcription Polymerase Chain Reaction, Expressing, Confocal Microscopy, Imaging, Cotransfection, Construct, Activation Assay, Quantitative RT-PCR, In Silico, Binding Assay, Incubation, In Vitro, Labeling, Transfection, Concentration Assay, Over Expression

Journal: PLoS ONE

Article Title: Zip4 (Slc39a4) Expression is Activated in Hepatocellular Carcinomas and Functions to Repress Apoptosis, Enhance Cell Cycle and Increase Migration

doi: 10.1371/journal.pone.0013158

Figure Lengend Snippet: Immunofluorescence detection of ZIP4 in human HCC. Part A: Frozen sections of HCC from patients 3, 4 and 11 and normal liver tissue from patient 3 (see Table 1 ) were fixed, permeablized and blocked then ZIP4 was detected using an antipeptide antibody against mouse ZIP4 [26] , [47] . The peptide immunogen is well conserved between mouse and human and specificity of this antibody has been established [26] , [27] , [46] . Sections were washed and incubated sequentially with biotinylated secondary antibody then QDot 655 streptavidin conjugate. Red indicates the presence of ZIP4 whereas blue indicates nuclei stained with DAPI. Adobe Photoshop image capture software was used to capture these images. Part B: Frozen serial sections from patients 3 4 stained with hematoxylin and eosin to reveal the cellular structure of the ZIP4 positive tumors. Arrows point to the boundary of well-circumscribed lesions containing ZIP4 positive hepatocytes.

Article Snippet: Section s were washed with PBS, incubated with biotinylated secondary antibody followed by streptavidin-conjugated horseradish peroxidase and then developed in diaminobenzidine.

Techniques: Immunofluorescence, Incubation, Staining, Software

Journal: Molecular Therapy: the Journal of the American Society of Gene Therapy

Article Title: Mimicking Aspects of Frontotemporal Lobar Degeneration and Lou Gehrig's Disease in Rats via TDP-43 Overexpression

doi: 10.1038/mt.2009.3

Figure Lengend Snippet: Labeling for apoptotic nuclei. End labeling with biotinylated nucleotides was visualized with a diaminobenzidine chromagen with a methylene green counterstain. End labeling occurred after ( a ) TDP-43 injections, but not after ( b ) control injections ( c ) higher magnification of end labeling as in a . Interval of 2 weeks and equal vector doses of 3 × 10 10 vector genomes. a,b , bar = 34 µm; c , bar = 21 µm.

Article Snippet: Biotinylated secondary antibodies for peroxidase staining were from DAKO Cytomation (1:2,000; Carpinteria, CA), incubated for 1 hour at room temperature.

Techniques: Labeling, End Labeling, Plasmid Preparation

Journal: Development (Cambridge, England)

Article Title: Frizzled-5, a receptor for the synaptic organizer Wnt7a, regulates activity-mediated synaptogenesis

doi: 10.1242/dev.046722

Figure Lengend Snippet: Fz5CRD abolishes the effect of HFS on the localization of Fz5 at the surface and the formation of synapses. ( A ) Representative blots showing the effect of Fz5CRD on sFz5 in stimulated and non-stimulated neurons. sFz5, biotinylated sFz5; Fz5; total Fz5.

Article Snippet: Cells were fixed with 4% PFA and 4% sucrose in PBS, and incubated with primary antibodies to HA and Myc followed by incubation with biotinylated secondary antibodies (Amersham), Alexa 488 conjugated secondary antibodies and Hoechst (Molecular Probes).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: WNK2 Kinase Is a Novel Regulator of Essential Neuronal Cation-Chloride Cotransporters *

doi: 10.1074/jbc.M111.222893

Figure Lengend Snippet: Effect of WNK2 and WNK3 on the protein level and surface expression of NKCC1 and KCC4. Western blot analysis of the total and biotinylated fraction of proteins extracted from oocytes injected with NKCC1 ( A ) or KCC4 ( B ) cRNA in the absence or presence of WNK2 or WNK3 cRNA. C and D depict the results of the functional expression performed the same day using oocytes from the same batch for NKCC1 or KCC4, respectively, expressed as bumetanide-sensitive ( C ) or chloride-dependent ( D ) 86 Rb + uptake.

Article Snippet: Following washes with PBS, they were incubated with biotinylated secondary antibodies (Jackson Laboratories, 1:250 dilution) for 2 h at room temperature, washed in PBS, and then incubated with Vectastain ABC Elite solution (Vector Laboratories, Burlingame, CA) for 2 h. Sections were developed using 0.05% DAB (pH 7.4), 0.2% glucose, 0.01% nickel ammonium sulfate, 0.04% ammonium chloride, and 8 mg/ml glucose oxidase, and then rinsed, mounted onto glass slides, allowed to dry, dehydrated, and coverslipped.

Techniques: Expressing, Western Blot, Injection, Functional Assay

Journal: Veterinary World

Article Title: Detection of Toxoplasma gondii in cat’s internal organs by immunohistochemistry methods labeled with-[strept] avidin-biotin

doi: 10.14202/vetworld.2017.1035-1039

Figure Lengend Snippet: Toxoplasma positive immunohistochemistry staining of cat with labeled-(Strept) avidin-biotin method: (a) Necrosis is present in kidney and (b) ileum (arrow) (brown color is indicating the positive result of immunohistochemistry) (scale bar=30 µm).

Article Snippet: Slides were incubated with primary polyclonal anti-T. gondii antibody (rabbit) primary antibody (Abcam, USA) diluted 1/100 at 37°C for 32 min and biotinylated secondary antibody (Abcam, USA).

Techniques: Immunohistochemistry, Staining, Labeling, Avidin-Biotin Assay

Journal: Cell Death & Disease

Article Title: Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation

doi: 10.1038/cddis.2014.444

Figure Lengend Snippet: Cox-2 expression and interaction with Col10a1 cis-enhancer. ( a ) IHC assay using Cox-2 antibody on sagittal sections of mouse limb (fibula) at E17.5 showed that Cox-2 is strongly expressed in nuclei of hypertrophic chondrocytes but not in resting or proliferative chondrocytes (black box and arrows). Bottom showed higher magnification of the boxed area. Left panel shows no antibody control. ( b ) IHC assay using Cox-2 antibody on tibia sections at 4 weeks' age also detected high-level Cox-2 expression in nuclei of hypertrophic chondrocytes (left panel, black arrows). Right panel shows IHC assay of collagen X, which is mostly expressed extracellular within hypertrophic zone (red arrows). ( c ) EMSA assay detected specific binding complex (black arrow) formed by the Col10a1 enhancer and hypertrophic MCT cell nuclear extracts as previously described. 38 The signal intensity decreased when 200 ng of Cox-2 antibody was added, 500 ng of antibody further reduced the signal, whereas 1000 ng of antibody only showed faint signal (left panel). No signal reduction was seen in parallel experiment in which gradient amount of Nedd4 antibody was used (right panel). Data of non-biotinylated competitor control were not shown. ( d ) Illustrated is the result of ChIP experiment showing precipitated DNA enriched by Cox-2 antibody or control IgG. qPCR using primers flanking the enhancer suggested a significant enrichment (~7-fold, P =0.034) of the enhancer by Cox-2 antibody, whereas sequence flanking the intronic control region did not show significant enrichment (~3-fold, P =0.062)

Article Snippet: After washing with the 1xTBST (Tris Buffered Saline with 0.1% Tween-20), the slides were further incubated with biotinylated secondary antibody (anti-rabbit IgG, Santa Cruz) and detected using the ABC kit (Elite PK-6200 Universal, VECTOR Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Immunohistochemistry, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

Journal: Journal of Clinical Investigation

Article Title: Inhibition of endogenous thioredoxin in the heart increases oxidative stress and cardiac hypertrophy

doi: 10.1172/JCI200317700

Figure Lengend Snippet: The activities of the Ras–Raf-1–ERK pathway, p38-MAPK, and p46/p54–JNK1 were examined in Tg-DN-Trx1 mice and NTg littermates. ( a ) Some mice were subjected to aortic banding for 2 weeks. Activities of p42/p44–ERK ( a ), Raf-1 ( b ), p38-MAPK ( e ), and p46/p54–JNK1 ( f ) were determined using anti-phosphospecific Ab’s. The same filter was reprobed with respective non-phosphospecific Ab. ( e ) Cell extracts prepared from cardiac myocytes overexpressing mammalian sterile 20–like kinase 1 (Mst1) were used as positive control (P/C). The gel picture is representative of three to ten experiments in each immunoblot analysis. ( c ) The activity of Ras was determined using the Ras-binding domain in Raf-1 coupled with agarose. ( d ) S-thiolation of Ras was determined as described in Methods. COS-7 cells grown in 60-mm dishes were transfected with the indicated expression plasmids (2 μg). Cells were incubated with biotinylated cysteine (0.5 mM), and S-thiolated proteins were isolated by streptavidin-Sepharose. Samples were subjected to immunoblot analyses with anti-Ras Ab. AS-Trx1, antisense Trx1.

Article Snippet: After washing, biotinylated secondary Ab (anti-mouse IgG; BD Pharmingen) was applied for 1 hour, followed by streptavidin-HRP (BD Pharmingen) for 30 minutes at room temperature.

Techniques: Mouse Assay, Positive Control, Activity Assay, Binding Assay, Transfection, Expressing, Incubation, Isolation

Journal: Nature neuroscience

Article Title: Unique functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1

doi: 10.1038/nn.2837

Figure Lengend Snippet: Neto1 modulates kainate receptor function in the hippocampus. ( a-c ) Representative examples of inward currents elicited by agonist (3 μM kainate) from Neto1-knockout and wild-type littermate mice recorded in the whole-cell voltage clamp configuration in CA3 pyramidal cells ( a ), CA1 s. radiatum interneurons ( b ), and cerebellar Purkinje cells ( c ). Kainate-evoked inward currents were recorded in the presence of (in μM) 30 GYKI 53655, 50 d-APV, 100 picrotoxin, and 0.5 TTX. Each bar shows the mean peak current amplitude in each neuron from the numbers of cells;animals indicated. ( d ) To compare cell surface expression of KARs, acute hippocampal slices were prepared and biotinylated with cell-impermeable Sulfo-NHS-SS-biotin. After solubilization, biotinylated proteins were precipitated with Neutravidin-beads to isolate proteins at the cell surface. Most GluK2/3 was detected in the “Surface” fraction, whereas a cytosolic protein, tubulin, was detected in the “Internal” fraction. ( e ) No obvious change in surface expression of GluK2/3 or GluK5 was observed in acute hippocampal slices from wild-type and Neto1-knockout mice (n = 6). Scale bars (a-c): 50 μm. Data are given as mean ± s.e.m.; * P

Article Snippet: Then sections were incubated successively with 10% normal goat serum for 20 min, primary antibodies (1 μg/ml) overnight, biotinylated secondary antibodies for 2 h and avidin-biotin-peroxidase complex for 1 h, using a Histofine SAB-PO(R) kit (Nichirei Corp.).

Techniques: Knock-Out, Mouse Assay, Expressing