gfp antibody Search Results


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  • 99
    Thermo Fisher gfp tag polyclonal antibody
    Dg controls postsynaptic Spectrin concentration and both Cora and Spectrin co-immunoprecipitate with Dg. Double staining for alpha-Spectrin (Magenta) and HRP (green)(1) in (A) control (yw CS ) larvae, (B) larvae expressing muscle dg -RNAi (dg-RNAi/+; 24B Gal4/dg-RNAi), (C) dg e01554 /dg 323 mutant larvae and (D) larvae overexpressing Dg-C isoform in the muscles (24B-Gal4/UAS-DgC). Single stainings for alpha-Spectrin are shown in (2). Scale bar is 10 µm. (E) Co-immunoprecipitation was performed with a <t>polyclonal</t> <t>anti-GFP</t> antibody on protein extracts from flies expressing Dg-C-GFP. S corresponds to the supernatant and P to the pellet. Cora and alpha-Spectrin co-immunoprecipitate with Dg-C-GFP, but not Shaggy (Sgg), a cytoplasmic protein kinase.
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti gfp antibodies
    Increasing the ratio of <t>δ</t> cDNA increased δ incorporation into surface GABA A Rs with a concomitant decrease in β 2 subunit incorporation. Scatter plots of the subunit stoichiometry of surface α 4 BBS β 2 BBS::EGFP δ BBS receptors expressed using various cDNA transfection ratios for <t>anti-GFP-immunopurified</t> ( A ) and anti-δ-immunopurified ( B ) receptors. The number of each α 4 ( * ), β 2 (■), and δ (◇) subunit per surface GABA A R are plotted for each cDNA ratio. Aside from the transfection ratio on the far right, the amount of α 4 and β 2 cDNA remained the same, and the ratio of δ cDNA was increased. The bars are the means ± S.E. C , the average number of subunits/anti-GFP immunopurified surface receptor were plotted versus the ratio of δ cDNA transfected for 2:1: X (α:β:δ) cDNA transfections. The data were fit using a one-phase association (δ) or decay (α 4 and β 2 ) equation using Prism v5.02 (GraphPad Software). The data points are the means ± S.E. from ≥3 experiments, except for 2:1:5 cDNA ratio, where n = 1, and for the 2:1:0.5 cDNA ratio, where n = 2. IP , immunoprecipitation.
    Anti Gfp Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti gfp antibody
    Ldo45, the product of an intergenic splicing event, is required for Pdr16 targeting to LDs. (A) A GAL1 promoter was genomically integrated 5′ to YMR147W or YMR148W and targeting of Pdr16-Cherry to LDs, visualized with BODIPY, was assessed by fluorescence microscopy. Pdr16-Cherry targeting was abolished in the presence of glucose (repression) in both strains but rescued by incubation with galactose (induction) for 4 h only in GAL1p-YMR147W cells. Bar, 5 µm. (B) Schematic representation of the YMR147W (blue) and YMR148W (green) loci. A splicing reaction gives rise to a YMR147W - YMR148W ) encoding Ldo45. That transcript corresponds to most of the YMR147W sequence (dark blue) excluding the last 90 nucleotides (light blue), 210 nucleotides of the annotated YMR148W promoter (dark gray), and the full YMR148W sequence (green). Ldo16 is the translation product of YMR148W . (C) Proteins from indicated cells were extracted and subjected to SDS-PAGE and Western blotting using <t>anti–GFP</t> antibodies. C-terminal GFP-tagging of YMR148W resulted in two bands corresponding to Ldo16-GFP and Ldo45-GFP. N-terminal tagging of YMR148W or YMR147W gives rise to only one tagged protein (GFP-Ldo16 or GFP-Ldo45, respectively). Simultaneous tagging of YMR147W with Cherry and YMR148W with GFP gives rise to a Cherry-Ldo45-GFP protein migrating ∼30 kD higher than GFP-Ldo45. Promoters used included an endogenous promoter, lanes 2 and 3; NOP1p , lane 4; and TEF2p , lanes 6 and 7. (D) cDNA plasmids encoding GFP-Ldo16 or GFP-Ldo45* (asterisk marking deletion of the YMR148W start codon) under control of a strong constitutive TEF2 promoter or empty vector (e.v.) were transformed into cells with a genomic ymr148w deletion (lacking both Ldo16 and Ldo45) and targeting of Pdr16-Cherry to LDs, visualized with MDH, was assessed by fluorescence microscopy. Ldo16 is dispensable for Pdr16 localization, but overexpression of both Ldo isoforms induces alterations of LD morphology. Bar, 5 µm.
    Anti Gfp Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti gfp
    B16F10 in vivo–derived fusion hybrids. ( A ) B16F10 (H2B-RFP) cells (5 × 10 4 cells) intradermally injected into <t>GFP-expressing</t> mice ( n = 12, two hybrid clones) were harvested at ~1.0 cm at study end point. ( B ) Fluorescence analyses of tumor sections for RFP (red) and GFP (green) reveal double-positive hybrids and phagocytosed cancer cells with different nuclear morphology. Scale bar, 25 μm. ( C ) B16F10 (H2B-RFP/Cre) cells injected (5 × 10 4 cells) into R26R-stop-YFP transgenic mice ( n = 8). ( D ) Representative FACS plot of hybrid and unfused cancer cells from a dissociated tumor, for example, hybrids (red box) and unfused (black box) cancer cells ( n = 6 single tumor analyses, n = 2 pooled tumor analyses, n = 13 mice). ( E ) Three hundred FACS-isolated cells were injected into wild-type secondary recipient mice ( n = 19 unfused, n = 19 hybrids) analyzed for tumor growth at 40 days, and ( F ) 3000 FACS-isolated cells were injected into syngeneic recipient mice ( n = 3 MC38 injected mice, black lines; n = 3 hybrid injected mice, red lines) and temporally monitored for growth. ( G ) B16F10 (H2B-RFP) or MФ–B16F10-derived hybrid cells tail vein–injected into wild-type mice ( n = 12 mice). Macroscopic view of lungs and H E of a tissue section. Quantification of tumor area. ( H ) Flow analyses of in vivo–derived B16F10 fusion hybrids from a primary tumor. RFP/GFP coexpressing cells analyzed for cell surface MФ identity. All boxes represent hybrid populations. Open box denote hybrids that have lost CD45 expression ( n = 6 mice each). ( I ) B16F10 <t>(fl-dsRed-fl-eGFP)</t> cells intradermally injected into LysM-Cre mice ( n = 4) were harvested at ~1 cm. Primary tumor or metastatic lung tumors stained with antibodies to GFP (green) and the tumor protein microphtalmia-associated transcription factor (MITF, red). Scale bar, 25 μm.
    Anti Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 7280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Abcam)
    99
    Abcam gfp
    Specialization of Sema3a + cardiomyocytes into the conduction system in the developing heart. ( a – f ) Z-stack images of <t>RFP</t> and <t>GFP</t> immunostaining on Sema3a-CreERT2; R26-tdTomato; Cx40-GFP heart sections. Tamoxifen was administered at E12.5 ( a , b ), E14.5 ( c , d ) and E18.5 ( e , f ). The hearts were collected at P7 and P21 for each group. YZ indicates signals from the dotted lines on the Z-stack images. Scale bars, 50 µm. Each image is representative of 5 individual samples. ( g ) Schematic figure showing Sema3a + cells (red) and Cx40 + cells (green) in the developing and adult heart.
    Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8593 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher anti gfp antibody
    NEXN-AS1 and NEXN suppress the TLR4/NF-κB pathway and inflammatory gene expression. ( A ) Cultured human vascular endothelial cells were transfected with either an NEXN-AS1 –expressing lentivirus (LV- NEXN-AS1 ), the lentivirus vector (LV vector), an NEXN siRNA, and/or a scramble (control) siRNA, then stimulated with lipopolysaccharides (1 μg/ml) for 12 hours, followed by enzyme-linked immunosorbent assay of MCP1, TNF-α, and IL-6, respectively. ( B ) Cultured human vascular endothelial cells were transfected with either LV- NEXN-AS1 , LV vector, an NEXN siRNA, scramble (control) siRNA, NEXN-AS1 shRNA, scramble (control) shRNA, an NEXN-expressing plasmid (pcDNA-NEXN), and/or the plasmid vector (pcDNA-vector), followed by immunoblotting analysis using an anti-TLR4 antibody and an antibody against the housekeeping protein β-actin as a loading control. Graphs show fold differences in TLR4 band intensity standardized against β-actin band intensity. ( C and D ) Cells expressing <t>FLAG-tagged</t> TLR4 and <t>GFP-tagged</t> TLR4 were transfected with either LV- NEXN-AS1 , LV vector, pcDNA-NEXN, or pcDNA-vector, and then stimulated with lipopolysaccharides (1 μg/ml) for 12 hours. Thereafter, cells were subjected to protein immunoprecipitation using anti-FLAG antibody, followed by immunoblotting analysis using either an anti-FLAG antibody or an anti-GFP antibody. Representative immunoblot images are shown. Column charts show fold differences in TLR4-GFP band intensity standardized against TLR4-Flag band intensity in 5 experiments. ( E ) Cultured human vascular endothelial cells were transfected with LV- NEXN-AS1 , LV vector, NEXN siRNA, a scramble (control) siRNA, pcDNA-NEXN, and/or pcDNA vector, followed by NF-κB activity assay. ( F ) Cultured human vascular endothelial cells were transfected with NEXN siRNA, TLR4 siRNA, and/or a scramble (control) siRNA, followed by enzyme-linked immunosorbent assay of TNF-α and IL-6, respectively. Data are represented as mean ± SD values from 5 independent experiments. * P
    Anti Gfp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 3303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology anti gfp antibody
    IFIXα1 interacts with <t>HDM2.</t> (A) HDM2 interacts with IFIXα1 and IFIXβ1. 293T cells were cotransfected with HDM2 (2.5 μg) and EGFP vector (vector) (2.5 μg), EGFP-tagged IFIXα1 (α1) (2.5 μg), or IFIXβ1 (β1) (2.5 μg). Forty-eight hours posttransfection, cell lysates (500 μg) were immunoprecipitated with an anti-HDM2 antibody, and Western blotting was performed using an <t>anti-GFP</t> or anti-HDM2 antibody. (B) A reciprocal experiment that used anti-GFP antibody for IP and Western blotting with anti-IFIX or anti-HDM2 antibodies. (C) IFIXα1 interacts with HDM2 in the IFIXα1 stable cell lines. Cell lysates (600 μg) isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) or the empty vector cells (V) were immunoprecipitated using anti-HDM2 antibody and analyzed by Western blotting with anti-IFIXα or anti-HDM2 antibody. (D) The HDM2(1-441) mutant interacts with IFIXα1. 293T cells were transfected with HDM2 or the HDM2(1-441) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (V), followed by IP with anti-HDM2 antibody and Western blotting with either anti-HDM2 or anti-GFP antibody. (E and F) Amino acid region 150 to 230 of HDM2 interacts with IFIXα1. 293T cells were cotransfected with HDM2 or the HDM2(Δ150-230) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (E) or FLAG-IFIXα1 (α1) or FLAG empty vector (F), followed by IP/Western blotting with the indicated antibodies. An arrowhead indicates the HDM2 band (F). The untransfected 293T cells served as controls (C). (G) A summary of IFIXα1 binding by HDM2 and the HDM2(Δ150-230) and HDM2(1-441) mutants. (H and I) The HIN domain of IFIXα1 interacts with HDM2. (H) 293T cells transfected with HDM2 and EGFP empty vector, EGFP-IFIXα1, EGFP-IFIX-N (N), or EGFP-IFIX-HIN (HIN), followed by IP with anti-GFP antibody (left panel) or anti-HDM2 antibody (right panel) and Western blotting with anti-HDM2 or anti-GFP antibody. Untransfected 293T cells served as controls. (I) A summary of HDM2 binding by IFIXα1, IFIXβ1, IFIX-N, and IFIX-HIN.
    Anti Gfp Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rabbit anti gfp antibody
    Par3 forms a complex with BACE1 and regulates its trafficking (a) Hippocampal neurons were transfected with indicated constructs. At DIV11 neurons were immunostained for <t>FLAG</t> (red) and TGN (green). Merge shows the overlap between BACE1 and TGN. Scale bar: 10μm. (b) Quantification of colocalization of BACE1 and TGN. (c) Hippocampal neurons were transfected with indicated constructs together with <t>hBACE1-GFP</t> and LAMP1-mRFP and imaged live on DIV11. Scale bar: 10μm. (d) Quantification of colocalization of hBACE1-GFP and LAMP1-mRFP. Data were expressed as Mean ± SEM with Student’s t test, *p
    Rabbit Anti Gfp Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc gfp
    Structure of <t>TCF-4-P2/3</t> mutants and analysis of TCF transcriptional activity after co-transfection with wild-type HIPK2 or a kinase-inactive mutant (A) Amino acid sequence of TCF-4J, TCF-4K, and TCF-4-P2/3 mutants (TCF-4J-P2/3 and TCF-4K-P2/3) showing substitution of (Ser 154 , Ser 156 , Thr 178 , and Thr 189 ) to alanine. Mutation sites are indicated by bold letters. (B-C) Expressions of TCF-4 and HIPK2 in total cell lysate (TCL) and nuclear extracts (Nuc) immunoprecipitated (IP) with anti-c-Myc antibody, and TCF transcriptional activity of TCF-4J, TCF-4K, and TCF-4-P2/3 mutants (TCF-4J-P2/3 and TCF-4K-P2/3) after co-transfection with wild-type HIPK2 (wt) or a kinase-inactive mutant (mt) in HAK-1A (B) and Huh7 (C) cells. Expression levels of TCF-4 and HIPK2 were evaluated by Western blot analysis using antibodies against Myc-tag (arrow) and <t>GFP,</t> respectively. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p
    Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gfp n terminal antibody
    Lead triggers phase separation of <t>TDP-43</t> and decreases its solubility. A, Purified TDP-43 LLPS in vitro is facilitated by lead (II) acetate trihydrate (Pb) in a dose-dependent manner. Representative 63× DIC images. Arrows: examples of single TDP-43 droplets. Asterisk: examples of amorphous TDP-43 consolidates. Arrowheads: high-contrast, inert 1 micron polystyrene microspheres added to aid sample focusing. Scale bar = 10 µm. B, LLPS was quantified using an ImageJ algorithm as percentage of ROI covered by droplets. Points at mean, with error bars at SEM, were fit by nonlinear regression analysis (line) and the LogEC50 calculated as 160 µM (dotted line). Induced PC12 cells accumulate insoluble TDP-43:: GFP upon treatment with lead (Pb). Immunoblots of RIPA insoluble (C), RIPA soluble (D) and total RIPA lysate (E) were probed with anti-GFP antibodies ( N = 3). F, Densitometric analysis of total TDP-43:: GFP/Actin bands (from panel E) show an increase in TDP-43:: GFP in response to 0.174 and 0.521 µM lead but an decrease in response to 4.69 µM lead. G, At 1.56 and 4.69 µM concentrations, lead significantly increases the ratio of insoluble TDP-43:: GFP to soluble TDP-43:: GFP. N = 3; mean ± SEM; ANOVA w/Dunnett’s multiple comparison test, * p
    Anti Gfp N Terminal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 864 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gfp tag monoclonal antibody
    Telomere fusions in <t>Z4</t> and HeT-A mutants. (A) Mitotic chromosome preparations of third instar larvae. Z4 7.1 /Z4 7.1 , Z4 2.1 /+ and pzg 66 /+ alleles show telomere fusions. woc 964/ woc B111 mutants were used as a positive control and w 1118 as negative control. Red arrowheads indicate telomere fusions. (B) Anaphase preparations from third instar larvae neuroblasts of the Z4 7.1 /Z4 7.1 , woc 964/ woc B111 alleles and w 1118 . Defective anaphases due to telomere fusions visible in Z4 7.1 /Z4 7.1 , woc 964/ woc B111 (positive control). Normal anaphases in w 1118 (negative control). (C) Mitotic S2 cells stained with DAPI after Z4 , HeT-A gag gene, cav (HOAP), and unspecific ( Sart1 ) RNAi treatment. Telomere fusions are observed in Z4 and HeT-A Gag RNAi mutants; RNAi for the cav and the Sart1 non-LTR retrotransposon from Bombyx mori were used as positive and negative control respectively. Red arrowheads indicate telomere fusions. (D) Percentage of telomere fusions found in mitotic S2 cells after RNAi treatment. (E) Mitotic chromosome preparations of <t>HOAP-GFP/HOAP-GFP</t> and Z4 7.1 /HOAP-GFP third instar larvae neuroblasts. Chromosomes stained with DAPI (blue) and HOAP-GFP fusion protein (green). HOAP is present in Z4 telomere fusions (red arrowheads). (F) Percentage of telomere fusions found in Z4 7.1 /ligIV and Z4 / JIL-1 double mutants. ligIV and JIL-1 mutants do not affect the number of telomere fusions in Z4 mutants. For each mutant a minimum of 100 metaphases/anaphases of three different preparations were analyzed.
    Gfp Tag Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 884 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam antibodies against gfp
    Generation of primary and secondary cardiospheres and their characteristics . ( a ) Timeline of primary CS, SDC, and secondary CS generation. Within 48 hours, secondary CSs were generated from SDCs. ( b ) Phase-contrast bright field images and step-by-step average cell numbers ( n = 5). Bar: 500 µm. ( c ) The gene expressions of <t>Oct4</t> and c-kit were measured by real-time PCR. ( d ) The protein expressions of Oct4 and c-kit were measured by western blots and quantified by densitometry. ( e ) Primary and secondary CSs were positive for alkaline phosphatase (ALP) staining, but SDCs were not. The protein expressions of Oct4 and c-kit were assessed by confocal imaging. Oct4 expression was verified using Oct4 promoter-driven <t>GFP</t> cells. Nuclear colocalization of Oct4 was also confirmed in the single, dissociated secondary CS cells ( Supplementary Figure S1 ). Secondary CSs expressed Oct4 and c-kit more homogeneously and densely than primary CSs, whereas SDCs lacked expressions. TO-PRO-3 (nuclei). Bar: 50 µm. AU, arbitrary unit; CS, cardiosphere; FN, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; PDL, poly-D-lysine; SDC, sphere-derived cell.
    Antibodies Against Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti green fluorescent protein gfp antibody
    Synaptic transmission regulates the size and density of <t>GFP-GLT-1</t> clusters
    Monoclonal Anti Green Fluorescent Protein Gfp Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 439 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gfp
    Bilirubin activates sensory neurons in an MRGPR-dependent manner. ( A–D ) Confocal microscopy immunofluorescence images of adult mouse tissue sections from Mrgpra1 <t>GFP</t> animals with GFP expression under the control of the endogenous Mrgpra1 locus. ( A ) Mrgpra1 expression in dorsal root ganglia. Green depicts Mrgpra1 GFP . Red depicts anti-PLAP antibody staining where PLAP expression is controlled by the endogenous Mrgprd locus ( Mrgprd PLAP ). Blue depicts antibody staining against calcitonin gene-related peptide (CGRP). Scale bar is 50 µM. ( B ) Trigeminal ganglia (TG) stained with Mrgpra1 GFP (green) and anti-Substance P antibody (red). Scale bar is 50 μm. ( C ) Back skin stained with anti-GFP antibody (green) to visualize Mrgpra1 GFP nerve fibers in the dermis. Blue represents DAPI counterstain. Scale bar is 50 μm. ( D ) Spinal cord (SC) (lamina 1 and 2) stained with anti-GFP and IB4-564. Mrgpra1 GFP (green) staining was found in lamina two along with IB4 (red) positive terminals. Scale bar is 100 μm. ( E ) Representative whole-cell current-clamp recording of either WT or A1 KO DRG neurons. In WT DRG, bilirubin elicited action potentials in 5 out of 50 small-diameter neurons. In A1 KO DRG, bilirubin elicited action potentials in 0 out of 60 small-diameter neurons. Fisher’s exact test p
    Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dg controls postsynaptic Spectrin concentration and both Cora and Spectrin co-immunoprecipitate with Dg. Double staining for alpha-Spectrin (Magenta) and HRP (green)(1) in (A) control (yw CS ) larvae, (B) larvae expressing muscle dg -RNAi (dg-RNAi/+; 24B Gal4/dg-RNAi), (C) dg e01554 /dg 323 mutant larvae and (D) larvae overexpressing Dg-C isoform in the muscles (24B-Gal4/UAS-DgC). Single stainings for alpha-Spectrin are shown in (2). Scale bar is 10 µm. (E) Co-immunoprecipitation was performed with a polyclonal anti-GFP antibody on protein extracts from flies expressing Dg-C-GFP. S corresponds to the supernatant and P to the pellet. Cora and alpha-Spectrin co-immunoprecipitate with Dg-C-GFP, but not Shaggy (Sgg), a cytoplasmic protein kinase.

    Journal: PLoS ONE

    Article Title: Muscle Dystroglycan Organizes the Postsynapse and Regulates Presynaptic Neurotransmitter Release at the Drosophila Neuromuscular Junction

    doi: 10.1371/journal.pone.0002084

    Figure Lengend Snippet: Dg controls postsynaptic Spectrin concentration and both Cora and Spectrin co-immunoprecipitate with Dg. Double staining for alpha-Spectrin (Magenta) and HRP (green)(1) in (A) control (yw CS ) larvae, (B) larvae expressing muscle dg -RNAi (dg-RNAi/+; 24B Gal4/dg-RNAi), (C) dg e01554 /dg 323 mutant larvae and (D) larvae overexpressing Dg-C isoform in the muscles (24B-Gal4/UAS-DgC). Single stainings for alpha-Spectrin are shown in (2). Scale bar is 10 µm. (E) Co-immunoprecipitation was performed with a polyclonal anti-GFP antibody on protein extracts from flies expressing Dg-C-GFP. S corresponds to the supernatant and P to the pellet. Cora and alpha-Spectrin co-immunoprecipitate with Dg-C-GFP, but not Shaggy (Sgg), a cytoplasmic protein kinase.

    Article Snippet: The supernatant was incubated with anti-GFP antibody (A6455, Molecular Probes) O/N at 4°C.

    Techniques: Concentration Assay, Double Staining, Expressing, Mutagenesis, Immunoprecipitation

    Increasing the ratio of δ cDNA increased δ incorporation into surface GABA A Rs with a concomitant decrease in β 2 subunit incorporation. Scatter plots of the subunit stoichiometry of surface α 4 BBS β 2 BBS::EGFP δ BBS receptors expressed using various cDNA transfection ratios for anti-GFP-immunopurified ( A ) and anti-δ-immunopurified ( B ) receptors. The number of each α 4 ( * ), β 2 (■), and δ (◇) subunit per surface GABA A R are plotted for each cDNA ratio. Aside from the transfection ratio on the far right, the amount of α 4 and β 2 cDNA remained the same, and the ratio of δ cDNA was increased. The bars are the means ± S.E. C , the average number of subunits/anti-GFP immunopurified surface receptor were plotted versus the ratio of δ cDNA transfected for 2:1: X (α:β:δ) cDNA transfections. The data were fit using a one-phase association (δ) or decay (α 4 and β 2 ) equation using Prism v5.02 (GraphPad Software). The data points are the means ± S.E. from ≥3 experiments, except for 2:1:5 cDNA ratio, where n = 1, and for the 2:1:0.5 cDNA ratio, where n = 2. IP , immunoprecipitation.

    Journal: The Journal of Biological Chemistry

    Article Title: Stoichiometry of Expressed ?4?2? ?-Aminobutyric Acid Type A Receptors Depends on the Ratio of Subunit cDNA Transfected *

    doi: 10.1074/jbc.M110.104257

    Figure Lengend Snippet: Increasing the ratio of δ cDNA increased δ incorporation into surface GABA A Rs with a concomitant decrease in β 2 subunit incorporation. Scatter plots of the subunit stoichiometry of surface α 4 BBS β 2 BBS::EGFP δ BBS receptors expressed using various cDNA transfection ratios for anti-GFP-immunopurified ( A ) and anti-δ-immunopurified ( B ) receptors. The number of each α 4 ( * ), β 2 (■), and δ (◇) subunit per surface GABA A R are plotted for each cDNA ratio. Aside from the transfection ratio on the far right, the amount of α 4 and β 2 cDNA remained the same, and the ratio of δ cDNA was increased. The bars are the means ± S.E. C , the average number of subunits/anti-GFP immunopurified surface receptor were plotted versus the ratio of δ cDNA transfected for 2:1: X (α:β:δ) cDNA transfections. The data were fit using a one-phase association (δ) or decay (α 4 and β 2 ) equation using Prism v5.02 (GraphPad Software). The data points are the means ± S.E. from ≥3 experiments, except for 2:1:5 cDNA ratio, where n = 1, and for the 2:1:0.5 cDNA ratio, where n = 2. IP , immunoprecipitation.

    Article Snippet: A 25-μl aliquot of lysate supernatant was removed for Western blotting, and the remaining cell lysate labeled with anti-δ or anti-GFP antibodies was incubated with 120 μl of 20% slurry of protein A-Sepharose or protein G-Sepharose (Sigma-Aldrich), respectively.

    Techniques: Transfection, Software, Immunoprecipitation

    Ldo45, the product of an intergenic splicing event, is required for Pdr16 targeting to LDs. (A) A GAL1 promoter was genomically integrated 5′ to YMR147W or YMR148W and targeting of Pdr16-Cherry to LDs, visualized with BODIPY, was assessed by fluorescence microscopy. Pdr16-Cherry targeting was abolished in the presence of glucose (repression) in both strains but rescued by incubation with galactose (induction) for 4 h only in GAL1p-YMR147W cells. Bar, 5 µm. (B) Schematic representation of the YMR147W (blue) and YMR148W (green) loci. A splicing reaction gives rise to a YMR147W - YMR148W ) encoding Ldo45. That transcript corresponds to most of the YMR147W sequence (dark blue) excluding the last 90 nucleotides (light blue), 210 nucleotides of the annotated YMR148W promoter (dark gray), and the full YMR148W sequence (green). Ldo16 is the translation product of YMR148W . (C) Proteins from indicated cells were extracted and subjected to SDS-PAGE and Western blotting using anti–GFP antibodies. C-terminal GFP-tagging of YMR148W resulted in two bands corresponding to Ldo16-GFP and Ldo45-GFP. N-terminal tagging of YMR148W or YMR147W gives rise to only one tagged protein (GFP-Ldo16 or GFP-Ldo45, respectively). Simultaneous tagging of YMR147W with Cherry and YMR148W with GFP gives rise to a Cherry-Ldo45-GFP protein migrating ∼30 kD higher than GFP-Ldo45. Promoters used included an endogenous promoter, lanes 2 and 3; NOP1p , lane 4; and TEF2p , lanes 6 and 7. (D) cDNA plasmids encoding GFP-Ldo16 or GFP-Ldo45* (asterisk marking deletion of the YMR148W start codon) under control of a strong constitutive TEF2 promoter or empty vector (e.v.) were transformed into cells with a genomic ymr148w deletion (lacking both Ldo16 and Ldo45) and targeting of Pdr16-Cherry to LDs, visualized with MDH, was assessed by fluorescence microscopy. Ldo16 is dispensable for Pdr16 localization, but overexpression of both Ldo isoforms induces alterations of LD morphology. Bar, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation

    doi: 10.1083/jcb.201704122

    Figure Lengend Snippet: Ldo45, the product of an intergenic splicing event, is required for Pdr16 targeting to LDs. (A) A GAL1 promoter was genomically integrated 5′ to YMR147W or YMR148W and targeting of Pdr16-Cherry to LDs, visualized with BODIPY, was assessed by fluorescence microscopy. Pdr16-Cherry targeting was abolished in the presence of glucose (repression) in both strains but rescued by incubation with galactose (induction) for 4 h only in GAL1p-YMR147W cells. Bar, 5 µm. (B) Schematic representation of the YMR147W (blue) and YMR148W (green) loci. A splicing reaction gives rise to a YMR147W - YMR148W ) encoding Ldo45. That transcript corresponds to most of the YMR147W sequence (dark blue) excluding the last 90 nucleotides (light blue), 210 nucleotides of the annotated YMR148W promoter (dark gray), and the full YMR148W sequence (green). Ldo16 is the translation product of YMR148W . (C) Proteins from indicated cells were extracted and subjected to SDS-PAGE and Western blotting using anti–GFP antibodies. C-terminal GFP-tagging of YMR148W resulted in two bands corresponding to Ldo16-GFP and Ldo45-GFP. N-terminal tagging of YMR148W or YMR147W gives rise to only one tagged protein (GFP-Ldo16 or GFP-Ldo45, respectively). Simultaneous tagging of YMR147W with Cherry and YMR148W with GFP gives rise to a Cherry-Ldo45-GFP protein migrating ∼30 kD higher than GFP-Ldo45. Promoters used included an endogenous promoter, lanes 2 and 3; NOP1p , lane 4; and TEF2p , lanes 6 and 7. (D) cDNA plasmids encoding GFP-Ldo16 or GFP-Ldo45* (asterisk marking deletion of the YMR148W start codon) under control of a strong constitutive TEF2 promoter or empty vector (e.v.) were transformed into cells with a genomic ymr148w deletion (lacking both Ldo16 and Ldo45) and targeting of Pdr16-Cherry to LDs, visualized with MDH, was assessed by fluorescence microscopy. Ldo16 is dispensable for Pdr16 localization, but overexpression of both Ldo isoforms induces alterations of LD morphology. Bar, 5 µm.

    Article Snippet: Samples were analyzed by SDS-PAGE and Western blotting using an anti–GFP antibody (ChIP grade ab290; Abcam).

    Techniques: Fluorescence, Microscopy, Incubation, Sequencing, SDS Page, Western Blot, Plasmid Preparation, Transformation Assay, Over Expression

    Role of Ldo16 and Ldo45 in LD accumulation at the NVJ. (A) Overexpression of GFP-Ldo45 and GFP-Ldo16 leads to accumulation of LDs next to the NVJ, marked by Nvj1-Cherry (arrows; top). This effect was quantified by counting the number of LDs labeled by MDH that were close to the NVJ (black) and LDs that were far from the NVJ (gray; bottom). n = 200 LDs. Bar, 5 µm. (B) Overexpression of GFP-Ldo45 or GFP-Ldo16 results in the formation of LD clusters adjacent to vacuolar and nuclear membranes, compared with Ymr148w-GFP control cells expressing Ldo proteins from their endogenous promoters, as determined by immunoelectron microscopy using anti–GFP antibodies. Asterisks mark LDs. N, nucleus; V, vacuole. Bar, 250 nm.

    Journal: The Journal of Cell Biology

    Article Title: Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation

    doi: 10.1083/jcb.201704122

    Figure Lengend Snippet: Role of Ldo16 and Ldo45 in LD accumulation at the NVJ. (A) Overexpression of GFP-Ldo45 and GFP-Ldo16 leads to accumulation of LDs next to the NVJ, marked by Nvj1-Cherry (arrows; top). This effect was quantified by counting the number of LDs labeled by MDH that were close to the NVJ (black) and LDs that were far from the NVJ (gray; bottom). n = 200 LDs. Bar, 5 µm. (B) Overexpression of GFP-Ldo45 or GFP-Ldo16 results in the formation of LD clusters adjacent to vacuolar and nuclear membranes, compared with Ymr148w-GFP control cells expressing Ldo proteins from their endogenous promoters, as determined by immunoelectron microscopy using anti–GFP antibodies. Asterisks mark LDs. N, nucleus; V, vacuole. Bar, 250 nm.

    Article Snippet: Samples were analyzed by SDS-PAGE and Western blotting using an anti–GFP antibody (ChIP grade ab290; Abcam).

    Techniques: Over Expression, Labeling, Expressing, Immuno-Electron Microscopy

    Aberrant vesicular structures containing GFP–VAMP7-R150E are deficient in typical regulators of intracellular trafficking. (A–C) Transgenic X. laevis expressing the GFP–VAMP7-R150E mutant and examined by CLEM. Retinas were first examined by confocal microscopy (A) and then processed and analyzed by EM (B). Six cells in the confocal optical section (a–f) were matched to the cells (a–f) in the EM micrograph. The asterisk marks a control photoreceptor (f), not expressing GFP–VAMP7-R150E. The boxed area in B is magnified in C. m, mitochondria, Ly, lysosome. (D–F) Transgenic retinas expressing GFP–VAMP7-R150E fusion protein (green) labeled with anti-LAMP1 (r) (D), anti-GFP (r) (E), or anti-βCOP (r) (F). Arrows point to localization of the proteins examined. (G) PLA between: Rab6 (r) and anti-GFP (m) detecting GFP–VAMP7-R150E. (H,I) EM of the photoreceptor cell expressing the R150E mutant (H). The boxed area is magnified in I. Arrows point to vesicular structures with electron-dense content. (J–M) Transgenic GFP–VAMP7-R150E retinas labeled with anti-IRBP (m) (J), anti-ASAP1 (r) (K), anti-FIP3 (r) (L) and anti-VARP (r) (M). (N) PLA between syntaxin 3 (r) and anti-GFP (m). (O) Retinas labeled with anti-peripherin (m) (red). (P,Q) The f1 generation of GFP–VAMP7-R150E-expressing retinas stained with WGA (red). (R) Labeling with anti-LC3 (r) (red, arrows). (S) Epithelial cells outside of Xenopus eye labeled with anti-LC3 (r) (red, arrows point to autophagosomes). (T,U) Labeling with anti-ATG16L1 (m) (red, arrow) (T), or anti-ubiquitin (m) (U). Cells were visualized by DIC. (V) The f1 generation of GFP-VAMP7-Y45E-expressing retinas stained with WGA (red). Scale bar: 25 µm (for A); 5 µm (for B,D–H); 10 µm (for J–N); 1 µm (for C,I); 12 µm (for O,R,S,T,U); 20 µm (for Q); 50 µm (for P,V). M, myoid region; E, ellipsoid region; G, Golgi, N, nucleus; m, mouse antibody; r, rabbit antibody.

    Journal: Journal of Cell Science

    Article Title: An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

    doi: 10.1242/jcs.222034

    Figure Lengend Snippet: Aberrant vesicular structures containing GFP–VAMP7-R150E are deficient in typical regulators of intracellular trafficking. (A–C) Transgenic X. laevis expressing the GFP–VAMP7-R150E mutant and examined by CLEM. Retinas were first examined by confocal microscopy (A) and then processed and analyzed by EM (B). Six cells in the confocal optical section (a–f) were matched to the cells (a–f) in the EM micrograph. The asterisk marks a control photoreceptor (f), not expressing GFP–VAMP7-R150E. The boxed area in B is magnified in C. m, mitochondria, Ly, lysosome. (D–F) Transgenic retinas expressing GFP–VAMP7-R150E fusion protein (green) labeled with anti-LAMP1 (r) (D), anti-GFP (r) (E), or anti-βCOP (r) (F). Arrows point to localization of the proteins examined. (G) PLA between: Rab6 (r) and anti-GFP (m) detecting GFP–VAMP7-R150E. (H,I) EM of the photoreceptor cell expressing the R150E mutant (H). The boxed area is magnified in I. Arrows point to vesicular structures with electron-dense content. (J–M) Transgenic GFP–VAMP7-R150E retinas labeled with anti-IRBP (m) (J), anti-ASAP1 (r) (K), anti-FIP3 (r) (L) and anti-VARP (r) (M). (N) PLA between syntaxin 3 (r) and anti-GFP (m). (O) Retinas labeled with anti-peripherin (m) (red). (P,Q) The f1 generation of GFP–VAMP7-R150E-expressing retinas stained with WGA (red). (R) Labeling with anti-LC3 (r) (red, arrows). (S) Epithelial cells outside of Xenopus eye labeled with anti-LC3 (r) (red, arrows point to autophagosomes). (T,U) Labeling with anti-ATG16L1 (m) (red, arrow) (T), or anti-ubiquitin (m) (U). Cells were visualized by DIC. (V) The f1 generation of GFP-VAMP7-Y45E-expressing retinas stained with WGA (red). Scale bar: 25 µm (for A); 5 µm (for B,D–H); 10 µm (for J–N); 1 µm (for C,I); 12 µm (for O,R,S,T,U); 20 µm (for Q); 50 µm (for P,V). M, myoid region; E, ellipsoid region; G, Golgi, N, nucleus; m, mouse antibody; r, rabbit antibody.

    Article Snippet: Rabbit polyclonal antibodies were: anti-syntaxin 3 (110 033, Synaptic Systems, Germany), anti-ASAP1 ( ) (a gift from Paul Randazzo, National Cancer Institute, Bethesda, MD), anti-Arf4 , anti-Rab8 (R5530, Sigma), anti-Rabin8 ( ) (a gift from Johan Peranen, University of Helsinki, Finland), anti-phosphotyrosine (ab50722, Abcam), anti-Rab6 (sc-310, Santa Cruz Biotechnology), anti-VARP (24034-1-AP, Proteintech), anti-LAMP1 (ab24170, Abcam), anti-βCOP (G-2279, Sigma), anti-LC3 (ab128025, Abcam), anti-GST (G7781, Sigma), anti-6His (10001-0-AP, Proteintech), anti-GFP (ab290, Abcam); rabbit and goat anti-FIP3 antibodies, and FIP3F1 were gifts from Rytis Prekeris, University of Colorado, Denver, CO. Duolink in situ Detection Reagent Red (excitation, 594 nm; emission, 624 nm) was from Sigma-Aldrich (DUO92008).

    Techniques: Transgenic Assay, Expressing, Mutagenesis, Confocal Microscopy, Labeling, Proximity Ligation Assay, Staining, Whole Genome Amplification

    Optogenetic silencing of TrpV1-Cre + axon terminals in PB L selectively reduces face allodynia after capsaicin injection (a) Schematic illustration of intraperitoneal injection of a 1–2 day old TrpV1-Cre pup followed by a face and hindpaw von Frey tests in the same individual mice in TrpV1Cre::eArch (n = 9) and TrpV1Cre::GFP groups (n = 8). The order of face versus hindpaw tests was randomized. Each mouse was tested before and after 10 µL 4% capsaicin was injected into either face or hindpaw. (b) Representative post-hoc image of TrpV1Cre::eArch + axon terminals in PB L and labeled TrpV1Cre::eArch + cell bodies in TG (n = 9 mice; Scale bars, 50 µm). (c) Quantification of mechanical thresholds of face withdrawal responses in von Frey tests. Measurements were taken before and after capsaicin injection into right whisker pad, as well as without and with optogenetic silencing in TrpV1Cre::eArch (n = 9) or in control TrpV1Cre::GFP groups (n = 8, two-way repeated measures ANOVA; (eArch vs. GFP) P = > 0.9999, P = > 0.9999, P = > 0.9999, * P = 0.0440; (no light vs light) eArch: P = > 0.9999, ** P = 0.0046, GFP: P = > 0.9999, P = > 0.9999; F (3, 45) = 2.671). Data are mean ± SEM. (d) Quantification of mechanical thresholds of hindpaw withdrawal responses in von Frey tests. Measurements were taken before and after capsaicin injection into right hindpaw, as well as without and with optogenetic silencing in TrpV1Cre::eArch (n = 9) or in TrpV1Cre::GFP groups (n = 8, two-way repeated measures ANOVA; (Arch vs. GFP) P = > 0.9999, P = > 0.9999, P = > 0.9999, > 0.9999; (no light vs light) Arch: P = > 0.9999, P = > 0.9999, GFP: P = > 0.9999, P = > 0.9999; F (3, 45) = 0.03048). Data are mean ± SEM. (e) Schematic illustration of real-time place preference (RTPP) test of mouse injected with capsaicin into left whisker pad. (f) Quantification of time the experimental group spent in non-preferred chamber before capsaicin, after capsaicin, and without or with optogenetic silencing (n = 6 one-way repeated measures ANOVA; P = 0.5356, * P = 0.0174, ** P = 0.0031; F 2, 10 = 10.92). Data are mean ± SEM. (g) Quantification of time the control group spent in non-preferred chamber before capsaicin, after capsaicin, and without or with optogenetic silencing (n = 7; one-way repeated measures ANOVA; P = 0.7320, P = 0.2086, P = 0.5537; F 2, 10 = 1.695). Data are mean ± SEM.

    Journal: Nature neuroscience

    Article Title: A craniofacial-specific monosynaptic circuit enables heightened affective pain

    doi: 10.1038/s41593-017-0012-1

    Figure Lengend Snippet: Optogenetic silencing of TrpV1-Cre + axon terminals in PB L selectively reduces face allodynia after capsaicin injection (a) Schematic illustration of intraperitoneal injection of a 1–2 day old TrpV1-Cre pup followed by a face and hindpaw von Frey tests in the same individual mice in TrpV1Cre::eArch (n = 9) and TrpV1Cre::GFP groups (n = 8). The order of face versus hindpaw tests was randomized. Each mouse was tested before and after 10 µL 4% capsaicin was injected into either face or hindpaw. (b) Representative post-hoc image of TrpV1Cre::eArch + axon terminals in PB L and labeled TrpV1Cre::eArch + cell bodies in TG (n = 9 mice; Scale bars, 50 µm). (c) Quantification of mechanical thresholds of face withdrawal responses in von Frey tests. Measurements were taken before and after capsaicin injection into right whisker pad, as well as without and with optogenetic silencing in TrpV1Cre::eArch (n = 9) or in control TrpV1Cre::GFP groups (n = 8, two-way repeated measures ANOVA; (eArch vs. GFP) P = > 0.9999, P = > 0.9999, P = > 0.9999, * P = 0.0440; (no light vs light) eArch: P = > 0.9999, ** P = 0.0046, GFP: P = > 0.9999, P = > 0.9999; F (3, 45) = 2.671). Data are mean ± SEM. (d) Quantification of mechanical thresholds of hindpaw withdrawal responses in von Frey tests. Measurements were taken before and after capsaicin injection into right hindpaw, as well as without and with optogenetic silencing in TrpV1Cre::eArch (n = 9) or in TrpV1Cre::GFP groups (n = 8, two-way repeated measures ANOVA; (Arch vs. GFP) P = > 0.9999, P = > 0.9999, P = > 0.9999, > 0.9999; (no light vs light) Arch: P = > 0.9999, P = > 0.9999, GFP: P = > 0.9999, P = > 0.9999; F (3, 45) = 0.03048). Data are mean ± SEM. (e) Schematic illustration of real-time place preference (RTPP) test of mouse injected with capsaicin into left whisker pad. (f) Quantification of time the experimental group spent in non-preferred chamber before capsaicin, after capsaicin, and without or with optogenetic silencing (n = 6 one-way repeated measures ANOVA; P = 0.5356, * P = 0.0174, ** P = 0.0031; F 2, 10 = 10.92). Data are mean ± SEM. (g) Quantification of time the control group spent in non-preferred chamber before capsaicin, after capsaicin, and without or with optogenetic silencing (n = 7; one-way repeated measures ANOVA; P = 0.7320, P = 0.2086, P = 0.5537; F 2, 10 = 1.695). Data are mean ± SEM.

    Article Snippet: The primary antibodies used in this study are: goat anti-Fos (Santa Cruz Biotechnology, sc52-g, 1:300), rabbit anti-CGRP (Millipore, AB15360, 1:1000), sheep anti-FoxP2 (R & D Systems, AF5647, 1:5000), rabbit anti-NF200 (Sigma, N4142, 1:200), GS-IB4-Alexa 488–conjugated (Invitrogen, , 1:1000), rabbit anti-VR1(TrpV1) (Abcam, ab31895, 1:1000), and rabbit anti-GFP (Abcam, ab290, 1:1000).

    Techniques: Injection, Mouse Assay, Labeling, Whisker Assay

    Optogenetic activation of TrpV1-Cre + sensory axons activates PB L -nociceptive neurons and elicits aversive behavior and stress calls in a real-time place escape/avoidance task (a) Schematic illustration of intraperitoneal injection of a 1–2 day old TrpV1-Cre pup (n = 3), followed by optogenetic-assisted whole cell patch-clamp recording from a PB L neuron in acute brain slices (b) Representative traces from a cell showing no light-evoked IPSC at a holding potential of 10mV, but observed to have light-evoked EPSC at a holding of −65mV. Cell, held at −65 mV, was bath applied with 1µM TTX, followed by 100 µM 4-AP and 1µM TTX, showing a light-evoked monosynaptic EPSC. (c) Averaged current amplitude is shown. Data are mean ± SEM. (closed circles, individual cells, n = 15). (d) Representative high-mag image of TrpV1Cre::ChR2 + axon terminals and CANE-RV-mCherry captured PB L -pain neurons. (n = 3 mice; Scale bar, 50 µm). (e) Representative of an mCherry + PB L -pain neuron recorded to have light-evoked EPSC at a holding of −65mV. Cell was bath applied with 1µM TTX, followed by 100 µM 4-Ap and 1µM TTX, showing a light-evoked monosynaptic EPSC. (f) Averaged current amplitude is shown. Data are mean ± SEM. (closed circles, individual cells, n = 6). (g) Schematic illustration of real-time place escape/avoidance (PEA) test. (h) Representative spatial tracking map showing the location of an experimental mouse before, during, and after optogenetic stimulation of TrpV1Cre::ChR2 + axon terminals in the PB L in the preferred chamber. (i) Representative spatial tracking map showing the location of a control mouse before, during, and after illuminating TrpV1Cre::GFP + axon terminals in the PB L in the preferred chamber. (j) Percentage of preference (per 30 seconds) the experimental and control groups had before, during, and after optogenetic stimulation (n = 8 3) shown across time (min). Data are mean ± SEM. (k) Quantification of time theTrpV1Cre::ChR2 group spent in preferred chamber before, during, and after optogenetic stimulation (n = 8 one-way repeated measures ANOVA; *** *P =

    Journal: Nature neuroscience

    Article Title: A craniofacial-specific monosynaptic circuit enables heightened affective pain

    doi: 10.1038/s41593-017-0012-1

    Figure Lengend Snippet: Optogenetic activation of TrpV1-Cre + sensory axons activates PB L -nociceptive neurons and elicits aversive behavior and stress calls in a real-time place escape/avoidance task (a) Schematic illustration of intraperitoneal injection of a 1–2 day old TrpV1-Cre pup (n = 3), followed by optogenetic-assisted whole cell patch-clamp recording from a PB L neuron in acute brain slices (b) Representative traces from a cell showing no light-evoked IPSC at a holding potential of 10mV, but observed to have light-evoked EPSC at a holding of −65mV. Cell, held at −65 mV, was bath applied with 1µM TTX, followed by 100 µM 4-AP and 1µM TTX, showing a light-evoked monosynaptic EPSC. (c) Averaged current amplitude is shown. Data are mean ± SEM. (closed circles, individual cells, n = 15). (d) Representative high-mag image of TrpV1Cre::ChR2 + axon terminals and CANE-RV-mCherry captured PB L -pain neurons. (n = 3 mice; Scale bar, 50 µm). (e) Representative of an mCherry + PB L -pain neuron recorded to have light-evoked EPSC at a holding of −65mV. Cell was bath applied with 1µM TTX, followed by 100 µM 4-Ap and 1µM TTX, showing a light-evoked monosynaptic EPSC. (f) Averaged current amplitude is shown. Data are mean ± SEM. (closed circles, individual cells, n = 6). (g) Schematic illustration of real-time place escape/avoidance (PEA) test. (h) Representative spatial tracking map showing the location of an experimental mouse before, during, and after optogenetic stimulation of TrpV1Cre::ChR2 + axon terminals in the PB L in the preferred chamber. (i) Representative spatial tracking map showing the location of a control mouse before, during, and after illuminating TrpV1Cre::GFP + axon terminals in the PB L in the preferred chamber. (j) Percentage of preference (per 30 seconds) the experimental and control groups had before, during, and after optogenetic stimulation (n = 8 3) shown across time (min). Data are mean ± SEM. (k) Quantification of time theTrpV1Cre::ChR2 group spent in preferred chamber before, during, and after optogenetic stimulation (n = 8 one-way repeated measures ANOVA; *** *P =

    Article Snippet: The primary antibodies used in this study are: goat anti-Fos (Santa Cruz Biotechnology, sc52-g, 1:300), rabbit anti-CGRP (Millipore, AB15360, 1:1000), sheep anti-FoxP2 (R & D Systems, AF5647, 1:5000), rabbit anti-NF200 (Sigma, N4142, 1:200), GS-IB4-Alexa 488–conjugated (Invitrogen, , 1:1000), rabbit anti-VR1(TrpV1) (Abcam, ab31895, 1:1000), and rabbit anti-GFP (Abcam, ab290, 1:1000).

    Techniques: Activation Assay, Injection, Patch Clamp, Mouse Assay

    H2A.Z acidic patch is incorporated at lower levels at target genes. ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z WT , H2A.Z AP3 , H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z WT , H2A.Z AP3 , and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z WT , H2A.Z AP3 , and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z WT and H2A.Z AP3 were generated using GFP antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z WT and H2A.Z AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z WT in chromatin fractions compared to H2A.Z AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z WT and H2A.Z AP3 . Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z AP3 . (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z WT and H2A.Z AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z WT and H2A.Z AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.

    Journal: PLoS Genetics

    Article Title: H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

    doi: 10.1371/journal.pgen.1003725

    Figure Lengend Snippet: H2A.Z acidic patch is incorporated at lower levels at target genes. ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z WT , H2A.Z AP3 , H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z WT , H2A.Z AP3 , and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z WT , H2A.Z AP3 , and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z WT and H2A.Z AP3 were generated using GFP antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z WT and H2A.Z AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z WT in chromatin fractions compared to H2A.Z AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z WT and H2A.Z AP3 . Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z AP3 . (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z WT and H2A.Z AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z WT and H2A.Z AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.

    Article Snippet: Rabbit anti-H2A.Z antibody (Abcam, ab4174) and Rabbit anti-GFP antibody (Abcam, ab290) was used for western blot at concentrations recommended by the manufacturer.

    Techniques: Chromatin Immunoprecipitation, Expressing, Generated, RNA Sequencing Assay, Binding Assay, Western Blot, Immunoprecipitation, Isolation, SDS Page

    B16F10 in vivo–derived fusion hybrids. ( A ) B16F10 (H2B-RFP) cells (5 × 10 4 cells) intradermally injected into GFP-expressing mice ( n = 12, two hybrid clones) were harvested at ~1.0 cm at study end point. ( B ) Fluorescence analyses of tumor sections for RFP (red) and GFP (green) reveal double-positive hybrids and phagocytosed cancer cells with different nuclear morphology. Scale bar, 25 μm. ( C ) B16F10 (H2B-RFP/Cre) cells injected (5 × 10 4 cells) into R26R-stop-YFP transgenic mice ( n = 8). ( D ) Representative FACS plot of hybrid and unfused cancer cells from a dissociated tumor, for example, hybrids (red box) and unfused (black box) cancer cells ( n = 6 single tumor analyses, n = 2 pooled tumor analyses, n = 13 mice). ( E ) Three hundred FACS-isolated cells were injected into wild-type secondary recipient mice ( n = 19 unfused, n = 19 hybrids) analyzed for tumor growth at 40 days, and ( F ) 3000 FACS-isolated cells were injected into syngeneic recipient mice ( n = 3 MC38 injected mice, black lines; n = 3 hybrid injected mice, red lines) and temporally monitored for growth. ( G ) B16F10 (H2B-RFP) or MФ–B16F10-derived hybrid cells tail vein–injected into wild-type mice ( n = 12 mice). Macroscopic view of lungs and H E of a tissue section. Quantification of tumor area. ( H ) Flow analyses of in vivo–derived B16F10 fusion hybrids from a primary tumor. RFP/GFP coexpressing cells analyzed for cell surface MФ identity. All boxes represent hybrid populations. Open box denote hybrids that have lost CD45 expression ( n = 6 mice each). ( I ) B16F10 (fl-dsRed-fl-eGFP) cells intradermally injected into LysM-Cre mice ( n = 4) were harvested at ~1 cm. Primary tumor or metastatic lung tumors stained with antibodies to GFP (green) and the tumor protein microphtalmia-associated transcription factor (MITF, red). Scale bar, 25 μm.

    Journal: Science Advances

    Article Title: Cell fusion potentiates tumor heterogeneity and reveals circulating hybrid cells that correlate with stage and survival

    doi: 10.1126/sciadv.aat7828

    Figure Lengend Snippet: B16F10 in vivo–derived fusion hybrids. ( A ) B16F10 (H2B-RFP) cells (5 × 10 4 cells) intradermally injected into GFP-expressing mice ( n = 12, two hybrid clones) were harvested at ~1.0 cm at study end point. ( B ) Fluorescence analyses of tumor sections for RFP (red) and GFP (green) reveal double-positive hybrids and phagocytosed cancer cells with different nuclear morphology. Scale bar, 25 μm. ( C ) B16F10 (H2B-RFP/Cre) cells injected (5 × 10 4 cells) into R26R-stop-YFP transgenic mice ( n = 8). ( D ) Representative FACS plot of hybrid and unfused cancer cells from a dissociated tumor, for example, hybrids (red box) and unfused (black box) cancer cells ( n = 6 single tumor analyses, n = 2 pooled tumor analyses, n = 13 mice). ( E ) Three hundred FACS-isolated cells were injected into wild-type secondary recipient mice ( n = 19 unfused, n = 19 hybrids) analyzed for tumor growth at 40 days, and ( F ) 3000 FACS-isolated cells were injected into syngeneic recipient mice ( n = 3 MC38 injected mice, black lines; n = 3 hybrid injected mice, red lines) and temporally monitored for growth. ( G ) B16F10 (H2B-RFP) or MФ–B16F10-derived hybrid cells tail vein–injected into wild-type mice ( n = 12 mice). Macroscopic view of lungs and H E of a tissue section. Quantification of tumor area. ( H ) Flow analyses of in vivo–derived B16F10 fusion hybrids from a primary tumor. RFP/GFP coexpressing cells analyzed for cell surface MФ identity. All boxes represent hybrid populations. Open box denote hybrids that have lost CD45 expression ( n = 6 mice each). ( I ) B16F10 (fl-dsRed-fl-eGFP) cells intradermally injected into LysM-Cre mice ( n = 4) were harvested at ~1 cm. Primary tumor or metastatic lung tumors stained with antibodies to GFP (green) and the tumor protein microphtalmia-associated transcription factor (MITF, red). Scale bar, 25 μm.

    Article Snippet: Lung sections were cut to 8-μm thickness, baked for 30 min at 37°C, then subjected to antigen retrieval under standard conditions (R & D Systems, CTS016), blocked with DAKO Protein Block Serum-Free (Agilent, X090930-2), and incubated for 16 hours at 4°C with primary antibodies [anti-MITF (1:500; Abcam, ab12039), anti-dsRed (1:250; Clontech, 632496), and anti-GFP (1:1000; Abcam, ab13970)] in background-reducing antibody diluent (Agilent, S302281-2).

    Techniques: In Vivo, Derivative Assay, Injection, Expressing, Mouse Assay, Fluorescence, Transgenic Assay, FACS, Isolation, Flow Cytometry, Staining

    Degradation of Os WRKY11 protein by the UPS. a Rice ( Oryza sativa ) protoplasts were transformed with 35S:: OsWRKY11-YFP . After 4 h, protoplasts were treated with 100 μM MG132 and buffer for 1 day. Fluorescence was observed using a confocal laser scanning microscope. Scale bars: 30 μm. NT: non-treated control. b Immunoblot analysis of total protoplast proteins in the absence (−) or presence (+) of 100 μM MG132. Blots were probed with green fluorescent protein (GFP) antibody. The PAT antibody was used as a control for transformation efficiency

    Journal: Rice

    Article Title: Rice WRKY11 Plays a Role in Pathogen Defense and Drought Tolerance

    doi: 10.1186/s12284-018-0199-0

    Figure Lengend Snippet: Degradation of Os WRKY11 protein by the UPS. a Rice ( Oryza sativa ) protoplasts were transformed with 35S:: OsWRKY11-YFP . After 4 h, protoplasts were treated with 100 μM MG132 and buffer for 1 day. Fluorescence was observed using a confocal laser scanning microscope. Scale bars: 30 μm. NT: non-treated control. b Immunoblot analysis of total protoplast proteins in the absence (−) or presence (+) of 100 μM MG132. Blots were probed with green fluorescent protein (GFP) antibody. The PAT antibody was used as a control for transformation efficiency

    Article Snippet: Immunoblot analysis was performed with GFP antibody (Abcam; code: ab6556).

    Techniques: Transformation Assay, Fluorescence, Laser-Scanning Microscopy

    Simultaneous detection of vGlut2 and Gad65 in POMC neurons. A–C: POMC-EGFP was detected with a GFP antibody (A, red) after in situ hybridization for both vGlut2 using an FITC-labeled probe and TSA-Biotin detection (B, green) and Gad65 using DIG-labeled

    Journal: The Journal of comparative neurology

    Article Title: Expression of GABAergic and Glutamatergic Phenotypic Markers in Hypothalamic Proopiomelanocortin Neurons

    doi: 10.1002/cne.23127

    Figure Lengend Snippet: Simultaneous detection of vGlut2 and Gad65 in POMC neurons. A–C: POMC-EGFP was detected with a GFP antibody (A, red) after in situ hybridization for both vGlut2 using an FITC-labeled probe and TSA-Biotin detection (B, green) and Gad65 using DIG-labeled

    Article Snippet: This included the anti-FITC POD and anti-DIG AP antibodies, as well as a chicken-anti-GFP antibody at 4°C (1:2,000; Abcam, Boston, MA) to detect GFP when using the transgenic animals.

    Techniques: In Situ Hybridization, Labeling

    Pericyte recruitment during vascular remodelling. ( A ) Intravital imaging of the corneal vasculature of a 4-OHT treated Cdh5(PAC)-CreER T2 : R26R-eYFP:Ng2DsRED mouse 9 days post suturing. ECs are identified by YFP expression (green) and perivascular mural cells by expression of DsRED (red). Arterioles (labelled “A”) are extensively covered by mural cells while venules (labelled “V”) are not. ( B and B’) Immunofluorescence staining of a cornea at day 9 post suture implantation recapitulates the findings from live imaging. ECs and pericytes were labelled with antibodies against CD31 (blue) and NG2 (Red). ( C- C’) Intravital imaging of a cornea of a Pdgfrβ-GFP mouse at day 8 post suture implantation, showing PDGFRβ-expressing perivascular mural cells (GFP, white, red arrowheads) as well as non-vascular cells (C’, arrows). ( D ) Intravital confocal imaging of 4-OHT-induced Cdh5(PAC)-CreER T2 : R26R-eYFP mice visualising sprouting vessels (eYFP, green) with established lumens as indicated by the presence of the injected tracer (red) all the way to the tip position. ( E – H ) Intravital imaging of the expanding vascular plexus of Claudin5-GFP:Ng2DsRED mice from day 6 to day 9 post suturing, showing ECs (GFP, green) and mural cells (DsRED, red). Dashed line (white) indicates the border between the pericyte-covered and uncovered vasculature.

    Journal: Scientific Reports

    Article Title: Characterization of multi-cellular dynamics of angiogenesis and vascular remodelling by intravital imaging of the wounded mouse cornea

    doi: 10.1038/s41598-018-28770-7

    Figure Lengend Snippet: Pericyte recruitment during vascular remodelling. ( A ) Intravital imaging of the corneal vasculature of a 4-OHT treated Cdh5(PAC)-CreER T2 : R26R-eYFP:Ng2DsRED mouse 9 days post suturing. ECs are identified by YFP expression (green) and perivascular mural cells by expression of DsRED (red). Arterioles (labelled “A”) are extensively covered by mural cells while venules (labelled “V”) are not. ( B and B’) Immunofluorescence staining of a cornea at day 9 post suture implantation recapitulates the findings from live imaging. ECs and pericytes were labelled with antibodies against CD31 (blue) and NG2 (Red). ( C- C’) Intravital imaging of a cornea of a Pdgfrβ-GFP mouse at day 8 post suture implantation, showing PDGFRβ-expressing perivascular mural cells (GFP, white, red arrowheads) as well as non-vascular cells (C’, arrows). ( D ) Intravital confocal imaging of 4-OHT-induced Cdh5(PAC)-CreER T2 : R26R-eYFP mice visualising sprouting vessels (eYFP, green) with established lumens as indicated by the presence of the injected tracer (red) all the way to the tip position. ( E – H ) Intravital imaging of the expanding vascular plexus of Claudin5-GFP:Ng2DsRED mice from day 6 to day 9 post suturing, showing ECs (GFP, green) and mural cells (DsRED, red). Dashed line (white) indicates the border between the pericyte-covered and uncovered vasculature.

    Article Snippet: Primary antibodies used include goat anti CD31 (AF3628, R & D System), chicken anti GFP (ab13970, Abcam), mouse anti α-smooth muscle actin (α-SMA) (c6198, Sigma), rabbit anti PDGFRβ (ab32570, Abcam) and rabbit anti NG2 (ab5320, Millipore).

    Techniques: Imaging, Expressing, Immunofluorescence, Staining, Mouse Assay, Injection

    Specialization of Sema3a + cardiomyocytes into the conduction system in the developing heart. ( a – f ) Z-stack images of RFP and GFP immunostaining on Sema3a-CreERT2; R26-tdTomato; Cx40-GFP heart sections. Tamoxifen was administered at E12.5 ( a , b ), E14.5 ( c , d ) and E18.5 ( e , f ). The hearts were collected at P7 and P21 for each group. YZ indicates signals from the dotted lines on the Z-stack images. Scale bars, 50 µm. Each image is representative of 5 individual samples. ( g ) Schematic figure showing Sema3a + cells (red) and Cx40 + cells (green) in the developing and adult heart.

    Journal: Scientific Reports

    Article Title: Genetic targeting of Purkinje fibres by Sema3a-CreERT2

    doi: 10.1038/s41598-018-20829-9

    Figure Lengend Snippet: Specialization of Sema3a + cardiomyocytes into the conduction system in the developing heart. ( a – f ) Z-stack images of RFP and GFP immunostaining on Sema3a-CreERT2; R26-tdTomato; Cx40-GFP heart sections. Tamoxifen was administered at E12.5 ( a , b ), E14.5 ( c , d ) and E18.5 ( e , f ). The hearts were collected at P7 and P21 for each group. YZ indicates signals from the dotted lines on the Z-stack images. Scale bars, 50 µm. Each image is representative of 5 individual samples. ( g ) Schematic figure showing Sema3a + cells (red) and Cx40 + cells (green) in the developing and adult heart.

    Article Snippet: The primary antibodies and dilution ratios were as follows: RFP (Rockland, 600-401-379,1:500); ESR (Abcam, ab27595, prediluted); GFP (Nacalai Tesque, 04404–84, 1:100); GFP (Abcam, ab6662, 1:200); TNNI3 (Abcam, ab56357, 1:100); Cx40 (Alpha Diagnostic, Cx40-A, 1:100); HCN4 (Abcam, ab32675, 1:100); tyrosine hydroxylase (TH, Millipore, AB152, 1:100); and Tuj1 (Covance, MMS-435P, 1:100).

    Techniques: Immunostaining

    The adult expression map of Sema3a in the heart. ( a ) Schematic showing the crossing of the mice to generate the Sema3a-CreERT2; R26-tdTomato mice. ( b ) Genetic labelling of the Sema3a + cells via tamoxifen administration. ( c ) Whole-mount fluorescence and bright-field views of a Sema3a-CreERT2; R26-tdTomato mouse heart. ( d ) Immunostaining for RFP and TNNI3 on a Sema3a-CreERT2; R26-tdTomato heart section showing the rarity of RFP + cells in the atrium. LA, left atrium. ( e ) No Sema3a + cells were detected in the SA node. ( f ) The expression of Sema3a in the AV node. ( g ) Immunostaining for RFP, GFP and TNNI3 in a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart section showing that the CX40 + coronary artery (arrowhead) was negative for RFP. ( h ) Whole-mount fluorescence view of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart. LBB, left bundle branch; LPF, left Purkinje fibre; IVS, interventricular septum; LVW, left ventricular free wall. The dotted line indicates the limits between the IVS and the LVW. ( i ) Immunostaining for RFP and GFP on heart sections of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse. Sema3a was not detected in the LBB or RBB, which were positive for Cx40-GFP. ( j ) Z-stack confocal image showing that Sema3a was expressed in the Purkinje fibres. XZ and YZ indicate the signals from the dotted lines on the Z-stack images in ( j ). Scale bars, 1 mm in ( c ) 500 µm in ( e , g , h ) and 100 µm in ( d , f , i ) and ( j ). Each image is representative of 5 individual samples.

    Journal: Scientific Reports

    Article Title: Genetic targeting of Purkinje fibres by Sema3a-CreERT2

    doi: 10.1038/s41598-018-20829-9

    Figure Lengend Snippet: The adult expression map of Sema3a in the heart. ( a ) Schematic showing the crossing of the mice to generate the Sema3a-CreERT2; R26-tdTomato mice. ( b ) Genetic labelling of the Sema3a + cells via tamoxifen administration. ( c ) Whole-mount fluorescence and bright-field views of a Sema3a-CreERT2; R26-tdTomato mouse heart. ( d ) Immunostaining for RFP and TNNI3 on a Sema3a-CreERT2; R26-tdTomato heart section showing the rarity of RFP + cells in the atrium. LA, left atrium. ( e ) No Sema3a + cells were detected in the SA node. ( f ) The expression of Sema3a in the AV node. ( g ) Immunostaining for RFP, GFP and TNNI3 in a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart section showing that the CX40 + coronary artery (arrowhead) was negative for RFP. ( h ) Whole-mount fluorescence view of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart. LBB, left bundle branch; LPF, left Purkinje fibre; IVS, interventricular septum; LVW, left ventricular free wall. The dotted line indicates the limits between the IVS and the LVW. ( i ) Immunostaining for RFP and GFP on heart sections of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse. Sema3a was not detected in the LBB or RBB, which were positive for Cx40-GFP. ( j ) Z-stack confocal image showing that Sema3a was expressed in the Purkinje fibres. XZ and YZ indicate the signals from the dotted lines on the Z-stack images in ( j ). Scale bars, 1 mm in ( c ) 500 µm in ( e , g , h ) and 100 µm in ( d , f , i ) and ( j ). Each image is representative of 5 individual samples.

    Article Snippet: The primary antibodies and dilution ratios were as follows: RFP (Rockland, 600-401-379,1:500); ESR (Abcam, ab27595, prediluted); GFP (Nacalai Tesque, 04404–84, 1:100); GFP (Abcam, ab6662, 1:200); TNNI3 (Abcam, ab56357, 1:100); Cx40 (Alpha Diagnostic, Cx40-A, 1:100); HCN4 (Abcam, ab32675, 1:100); tyrosine hydroxylase (TH, Millipore, AB152, 1:100); and Tuj1 (Covance, MMS-435P, 1:100).

    Techniques: Expressing, Mouse Assay, Fluorescence, Immunostaining

    NEXN-AS1 and NEXN suppress the TLR4/NF-κB pathway and inflammatory gene expression. ( A ) Cultured human vascular endothelial cells were transfected with either an NEXN-AS1 –expressing lentivirus (LV- NEXN-AS1 ), the lentivirus vector (LV vector), an NEXN siRNA, and/or a scramble (control) siRNA, then stimulated with lipopolysaccharides (1 μg/ml) for 12 hours, followed by enzyme-linked immunosorbent assay of MCP1, TNF-α, and IL-6, respectively. ( B ) Cultured human vascular endothelial cells were transfected with either LV- NEXN-AS1 , LV vector, an NEXN siRNA, scramble (control) siRNA, NEXN-AS1 shRNA, scramble (control) shRNA, an NEXN-expressing plasmid (pcDNA-NEXN), and/or the plasmid vector (pcDNA-vector), followed by immunoblotting analysis using an anti-TLR4 antibody and an antibody against the housekeeping protein β-actin as a loading control. Graphs show fold differences in TLR4 band intensity standardized against β-actin band intensity. ( C and D ) Cells expressing FLAG-tagged TLR4 and GFP-tagged TLR4 were transfected with either LV- NEXN-AS1 , LV vector, pcDNA-NEXN, or pcDNA-vector, and then stimulated with lipopolysaccharides (1 μg/ml) for 12 hours. Thereafter, cells were subjected to protein immunoprecipitation using anti-FLAG antibody, followed by immunoblotting analysis using either an anti-FLAG antibody or an anti-GFP antibody. Representative immunoblot images are shown. Column charts show fold differences in TLR4-GFP band intensity standardized against TLR4-Flag band intensity in 5 experiments. ( E ) Cultured human vascular endothelial cells were transfected with LV- NEXN-AS1 , LV vector, NEXN siRNA, a scramble (control) siRNA, pcDNA-NEXN, and/or pcDNA vector, followed by NF-κB activity assay. ( F ) Cultured human vascular endothelial cells were transfected with NEXN siRNA, TLR4 siRNA, and/or a scramble (control) siRNA, followed by enzyme-linked immunosorbent assay of TNF-α and IL-6, respectively. Data are represented as mean ± SD values from 5 independent experiments. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Long noncoding RNA NEXN-AS1 mitigates atherosclerosis by regulating the actin-binding protein NEXN

    doi: 10.1172/JCI98230

    Figure Lengend Snippet: NEXN-AS1 and NEXN suppress the TLR4/NF-κB pathway and inflammatory gene expression. ( A ) Cultured human vascular endothelial cells were transfected with either an NEXN-AS1 –expressing lentivirus (LV- NEXN-AS1 ), the lentivirus vector (LV vector), an NEXN siRNA, and/or a scramble (control) siRNA, then stimulated with lipopolysaccharides (1 μg/ml) for 12 hours, followed by enzyme-linked immunosorbent assay of MCP1, TNF-α, and IL-6, respectively. ( B ) Cultured human vascular endothelial cells were transfected with either LV- NEXN-AS1 , LV vector, an NEXN siRNA, scramble (control) siRNA, NEXN-AS1 shRNA, scramble (control) shRNA, an NEXN-expressing plasmid (pcDNA-NEXN), and/or the plasmid vector (pcDNA-vector), followed by immunoblotting analysis using an anti-TLR4 antibody and an antibody against the housekeeping protein β-actin as a loading control. Graphs show fold differences in TLR4 band intensity standardized against β-actin band intensity. ( C and D ) Cells expressing FLAG-tagged TLR4 and GFP-tagged TLR4 were transfected with either LV- NEXN-AS1 , LV vector, pcDNA-NEXN, or pcDNA-vector, and then stimulated with lipopolysaccharides (1 μg/ml) for 12 hours. Thereafter, cells were subjected to protein immunoprecipitation using anti-FLAG antibody, followed by immunoblotting analysis using either an anti-FLAG antibody or an anti-GFP antibody. Representative immunoblot images are shown. Column charts show fold differences in TLR4-GFP band intensity standardized against TLR4-Flag band intensity in 5 experiments. ( E ) Cultured human vascular endothelial cells were transfected with LV- NEXN-AS1 , LV vector, NEXN siRNA, a scramble (control) siRNA, pcDNA-NEXN, and/or pcDNA vector, followed by NF-κB activity assay. ( F ) Cultured human vascular endothelial cells were transfected with NEXN siRNA, TLR4 siRNA, and/or a scramble (control) siRNA, followed by enzyme-linked immunosorbent assay of TNF-α and IL-6, respectively. Data are represented as mean ± SD values from 5 independent experiments. * P

    Article Snippet: The membrane was immunoblotted with an anti-FLAG antibody (Sigma-Aldrich, catalog F2555) and an anti-GFP antibody (Molecular Probes, catalog PA5-22688), respectively.

    Techniques: Expressing, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, shRNA, Immunoprecipitation, Activity Assay

    IFIXα1 interacts with HDM2. (A) HDM2 interacts with IFIXα1 and IFIXβ1. 293T cells were cotransfected with HDM2 (2.5 μg) and EGFP vector (vector) (2.5 μg), EGFP-tagged IFIXα1 (α1) (2.5 μg), or IFIXβ1 (β1) (2.5 μg). Forty-eight hours posttransfection, cell lysates (500 μg) were immunoprecipitated with an anti-HDM2 antibody, and Western blotting was performed using an anti-GFP or anti-HDM2 antibody. (B) A reciprocal experiment that used anti-GFP antibody for IP and Western blotting with anti-IFIX or anti-HDM2 antibodies. (C) IFIXα1 interacts with HDM2 in the IFIXα1 stable cell lines. Cell lysates (600 μg) isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) or the empty vector cells (V) were immunoprecipitated using anti-HDM2 antibody and analyzed by Western blotting with anti-IFIXα or anti-HDM2 antibody. (D) The HDM2(1-441) mutant interacts with IFIXα1. 293T cells were transfected with HDM2 or the HDM2(1-441) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (V), followed by IP with anti-HDM2 antibody and Western blotting with either anti-HDM2 or anti-GFP antibody. (E and F) Amino acid region 150 to 230 of HDM2 interacts with IFIXα1. 293T cells were cotransfected with HDM2 or the HDM2(Δ150-230) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (E) or FLAG-IFIXα1 (α1) or FLAG empty vector (F), followed by IP/Western blotting with the indicated antibodies. An arrowhead indicates the HDM2 band (F). The untransfected 293T cells served as controls (C). (G) A summary of IFIXα1 binding by HDM2 and the HDM2(Δ150-230) and HDM2(1-441) mutants. (H and I) The HIN domain of IFIXα1 interacts with HDM2. (H) 293T cells transfected with HDM2 and EGFP empty vector, EGFP-IFIXα1, EGFP-IFIX-N (N), or EGFP-IFIX-HIN (HIN), followed by IP with anti-GFP antibody (left panel) or anti-HDM2 antibody (right panel) and Western blotting with anti-HDM2 or anti-GFP antibody. Untransfected 293T cells served as controls. (I) A summary of HDM2 binding by IFIXα1, IFIXβ1, IFIX-N, and IFIX-HIN.

    Journal: Molecular and Cellular Biology

    Article Title: Interferon-Inducible Protein IFIX?1 Functions as a Negative Regulator of HDM2

    doi: 10.1128/MCB.26.5.1979-1996.2006

    Figure Lengend Snippet: IFIXα1 interacts with HDM2. (A) HDM2 interacts with IFIXα1 and IFIXβ1. 293T cells were cotransfected with HDM2 (2.5 μg) and EGFP vector (vector) (2.5 μg), EGFP-tagged IFIXα1 (α1) (2.5 μg), or IFIXβ1 (β1) (2.5 μg). Forty-eight hours posttransfection, cell lysates (500 μg) were immunoprecipitated with an anti-HDM2 antibody, and Western blotting was performed using an anti-GFP or anti-HDM2 antibody. (B) A reciprocal experiment that used anti-GFP antibody for IP and Western blotting with anti-IFIX or anti-HDM2 antibodies. (C) IFIXα1 interacts with HDM2 in the IFIXα1 stable cell lines. Cell lysates (600 μg) isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) or the empty vector cells (V) were immunoprecipitated using anti-HDM2 antibody and analyzed by Western blotting with anti-IFIXα or anti-HDM2 antibody. (D) The HDM2(1-441) mutant interacts with IFIXα1. 293T cells were transfected with HDM2 or the HDM2(1-441) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (V), followed by IP with anti-HDM2 antibody and Western blotting with either anti-HDM2 or anti-GFP antibody. (E and F) Amino acid region 150 to 230 of HDM2 interacts with IFIXα1. 293T cells were cotransfected with HDM2 or the HDM2(Δ150-230) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (E) or FLAG-IFIXα1 (α1) or FLAG empty vector (F), followed by IP/Western blotting with the indicated antibodies. An arrowhead indicates the HDM2 band (F). The untransfected 293T cells served as controls (C). (G) A summary of IFIXα1 binding by HDM2 and the HDM2(Δ150-230) and HDM2(1-441) mutants. (H and I) The HIN domain of IFIXα1 interacts with HDM2. (H) 293T cells transfected with HDM2 and EGFP empty vector, EGFP-IFIXα1, EGFP-IFIX-N (N), or EGFP-IFIX-HIN (HIN), followed by IP with anti-GFP antibody (left panel) or anti-HDM2 antibody (right panel) and Western blotting with anti-HDM2 or anti-GFP antibody. Untransfected 293T cells served as controls. (I) A summary of HDM2 binding by IFIXα1, IFIXβ1, IFIX-N, and IFIX-HIN.

    Article Snippet: To determine their ability to bind HDM2, we cotransfected 293T cells with HDM2 and EGFP, EGFP-tagged IFIXα1, IFIX-N, or IFIX-HIN, followed by IP using anti-GFP (Fig. , left panel) or anti-HDM2 (Fig. , right panel) antibody and Western blotting with either anti-HDM2 or anti-GFP antibody.

    Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Stable Transfection, Isolation, Mutagenesis, Transfection, Binding Assay

    mdm2 is required for p53 induction and nuclear accumulation by IFIXα1. (A) mdm2 is required for the increased p53 protein expression by IFIXα1. The p53 −/− MEF or DKO MEF was transfected with GFP-p53 (0.1 μg) and increasing amounts of IFIXα1 (0, 1, and 2 μg). Twenty-four hours posttransfection, cell lysates were analyzed by Western blotting using antibodies against p53, IFIXα1, and α-tubulin. (B) mdm2 is required for the p53-mediated transcriptional activation by IFIXα1. PG13-LUC (0.3 μg) was cotransfected with p53 (0.01 μg) with or without increasing amounts of IFIXα1 (0.845 μg and 1.69 μg) into the p53 −/− MEF or DKO MEF. pRL-TK (0.05 μg) was cotransfected to normalize transfection efficiency. Cells were harvested 24 h after transfection, and the luciferase activity was measured using a dual luciferase assay. The relative luciferase activity was obtained by setting the normalized activity of PG13-LUC alone at 1. (C) IFIXα1 promotes p53 nuclear localization. GFP-p53 was transfected into the IFIXα1 stable MCF-7 (X-1 and X-2) and the vector control (V) cells. Twenty-four hours after transfection, the number of cells in which GFP-p53 localized in both nucleus and cytoplasm (C+N) or predominantly in the nucleus (N) was counted. Average results were obtained from two independent experiments. V (C+N, 54.55% ± 3.45%; N, 45.45% ± 3.45%); X-1 (C+N, 36.1% ± 1.9%; N, 63.9% ± 1.9%); and X-2 (C+N, 21.15% ± 1.85%; N, 78.85% ± 1.85%). (D) IFIXα1 has little effect on p53 nuclear localization in the presence of the HDM2(C464A) mutant. GFP-p53 (0.25 μg) was cotransfected into the H1299 cells with HDM2 (0.75 μg) and IFIXα1 or empty vector (1.25 μg). Twenty-four hours after transfection, the number of cells in which GFP-p53 localized in both nucleus and cytoplasm or predominantly in the nucleus was counted. Average results were obtained from two independent experiments: V and HDM2 (C+N, 49% ± 3.4%; N, 51% ± 3.4%); IFIXα1 and HDM2 (C+N, 29% ± 0.4%; N, 71% ± 0.4%); V and C464A (C+N, 31.7% ± 3.1%; N, 68.3% ± 3.1%); and IFIXα1 and C464A (C+N, 27% ± 1.6%; N, 73% ± 1.6%). On average, more than 80 GFP-positive cells were counted in each transfection experiment.

    Journal: Molecular and Cellular Biology

    Article Title: Interferon-Inducible Protein IFIX?1 Functions as a Negative Regulator of HDM2

    doi: 10.1128/MCB.26.5.1979-1996.2006

    Figure Lengend Snippet: mdm2 is required for p53 induction and nuclear accumulation by IFIXα1. (A) mdm2 is required for the increased p53 protein expression by IFIXα1. The p53 −/− MEF or DKO MEF was transfected with GFP-p53 (0.1 μg) and increasing amounts of IFIXα1 (0, 1, and 2 μg). Twenty-four hours posttransfection, cell lysates were analyzed by Western blotting using antibodies against p53, IFIXα1, and α-tubulin. (B) mdm2 is required for the p53-mediated transcriptional activation by IFIXα1. PG13-LUC (0.3 μg) was cotransfected with p53 (0.01 μg) with or without increasing amounts of IFIXα1 (0.845 μg and 1.69 μg) into the p53 −/− MEF or DKO MEF. pRL-TK (0.05 μg) was cotransfected to normalize transfection efficiency. Cells were harvested 24 h after transfection, and the luciferase activity was measured using a dual luciferase assay. The relative luciferase activity was obtained by setting the normalized activity of PG13-LUC alone at 1. (C) IFIXα1 promotes p53 nuclear localization. GFP-p53 was transfected into the IFIXα1 stable MCF-7 (X-1 and X-2) and the vector control (V) cells. Twenty-four hours after transfection, the number of cells in which GFP-p53 localized in both nucleus and cytoplasm (C+N) or predominantly in the nucleus (N) was counted. Average results were obtained from two independent experiments. V (C+N, 54.55% ± 3.45%; N, 45.45% ± 3.45%); X-1 (C+N, 36.1% ± 1.9%; N, 63.9% ± 1.9%); and X-2 (C+N, 21.15% ± 1.85%; N, 78.85% ± 1.85%). (D) IFIXα1 has little effect on p53 nuclear localization in the presence of the HDM2(C464A) mutant. GFP-p53 (0.25 μg) was cotransfected into the H1299 cells with HDM2 (0.75 μg) and IFIXα1 or empty vector (1.25 μg). Twenty-four hours after transfection, the number of cells in which GFP-p53 localized in both nucleus and cytoplasm or predominantly in the nucleus was counted. Average results were obtained from two independent experiments: V and HDM2 (C+N, 49% ± 3.4%; N, 51% ± 3.4%); IFIXα1 and HDM2 (C+N, 29% ± 0.4%; N, 71% ± 0.4%); V and C464A (C+N, 31.7% ± 3.1%; N, 68.3% ± 3.1%); and IFIXα1 and C464A (C+N, 27% ± 1.6%; N, 73% ± 1.6%). On average, more than 80 GFP-positive cells were counted in each transfection experiment.

    Article Snippet: To determine their ability to bind HDM2, we cotransfected 293T cells with HDM2 and EGFP, EGFP-tagged IFIXα1, IFIX-N, or IFIX-HIN, followed by IP using anti-GFP (Fig. , left panel) or anti-HDM2 (Fig. , right panel) antibody and Western blotting with either anti-HDM2 or anti-GFP antibody.

    Techniques: Expressing, Transfection, Western Blot, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis

    IFIXα1 stabilizes p53 protein. (A) IFIXα1 exerts different effects on the p53 and HDM2 levels in p53-expressing cells. Total cell lysates isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) and the vector control (V) cell lines were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (B) The p53 status influences the IFIXα1 effect on HDM2 levels. HCT116 and HCT116(p53 −/− ) cells were transfected with EGFP vector or EGFP-IFIXα1. Forty-eight hours after transfection, the GFP-positive cells were collected using FACS. Cell lysates were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (C) IFIXα1 induces the steady-state HDM2 mRNA level but has little effect on p53 mRNA levels in MCF-7 cells. Total RNA (10 μg) isolated from the parental MCF-7 (C) and the stable cell lines transfected with the empty vector (V) or IFIXα1 expression vector (X-1 and X-2) was analyzed by Northern blotting using HDM2, p53, or IFIXα1 cDNA as a probe. The 18S and 28S rRNAs are shown as loading controls. (D) IFIXα1 increases p53 protein stability. The IFIXα1 stable MCF-7 (X-1 and X-2) and the vector control (V) cells were treated with CHX (100 μg /ml) for the time indicated. Cell lysates were analyzed for the expression of p53 and α-tubulin. (E and F) Depletion of IFIXα1 reduces p53 and p21 CIP1 expression levels. The IFIXα1 stable MCF-7 cell line, X-1, was transfected with siRNA specific to IFIXα (IFIX) (100 nM) or NS siRNA (100 nM). Forty-eight hours after transfection, the expression levels of p53, IFIXα1, p21 CIP1 , and α-tubulin were analyzed by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Interferon-Inducible Protein IFIX?1 Functions as a Negative Regulator of HDM2

    doi: 10.1128/MCB.26.5.1979-1996.2006

    Figure Lengend Snippet: IFIXα1 stabilizes p53 protein. (A) IFIXα1 exerts different effects on the p53 and HDM2 levels in p53-expressing cells. Total cell lysates isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) and the vector control (V) cell lines were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (B) The p53 status influences the IFIXα1 effect on HDM2 levels. HCT116 and HCT116(p53 −/− ) cells were transfected with EGFP vector or EGFP-IFIXα1. Forty-eight hours after transfection, the GFP-positive cells were collected using FACS. Cell lysates were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (C) IFIXα1 induces the steady-state HDM2 mRNA level but has little effect on p53 mRNA levels in MCF-7 cells. Total RNA (10 μg) isolated from the parental MCF-7 (C) and the stable cell lines transfected with the empty vector (V) or IFIXα1 expression vector (X-1 and X-2) was analyzed by Northern blotting using HDM2, p53, or IFIXα1 cDNA as a probe. The 18S and 28S rRNAs are shown as loading controls. (D) IFIXα1 increases p53 protein stability. The IFIXα1 stable MCF-7 (X-1 and X-2) and the vector control (V) cells were treated with CHX (100 μg /ml) for the time indicated. Cell lysates were analyzed for the expression of p53 and α-tubulin. (E and F) Depletion of IFIXα1 reduces p53 and p21 CIP1 expression levels. The IFIXα1 stable MCF-7 cell line, X-1, was transfected with siRNA specific to IFIXα (IFIX) (100 nM) or NS siRNA (100 nM). Forty-eight hours after transfection, the expression levels of p53, IFIXα1, p21 CIP1 , and α-tubulin were analyzed by Western blotting.

    Article Snippet: To determine their ability to bind HDM2, we cotransfected 293T cells with HDM2 and EGFP, EGFP-tagged IFIXα1, IFIX-N, or IFIX-HIN, followed by IP using anti-GFP (Fig. , left panel) or anti-HDM2 (Fig. , right panel) antibody and Western blotting with either anti-HDM2 or anti-GFP antibody.

    Techniques: Expressing, Isolation, Plasmid Preparation, Western Blot, Transfection, FACS, Stable Transfection, Northern Blot

    IFIX-HIN is sufficient to downregulate HDM2. (A) IFIX-HIN downregulates HDM2 expression. 293T cells were transfected with EGFP empty vector (EGFP) or EGFP-IFIX-HIN. MG132 (10 μM) treatment started at 5 h before harvest. Cell lysates isolated from the GFP-positive cells were analyzed by Western blotting using antibodies against HDM2, EGFP, and α-tubulin. (B to D) IFIX-HIN induces p53 and p21 CIP1 . H1299 cells were transfected with p53 (0.1 μg) and increasing amounts (0.5, 1.0, and 1.8 μg) of the FLAG-tagged IFIXα1 (B), IFIX-HIN (C), or IFIX-N (D), followed by Western blotting using antibodies against p53, IFIXα (B), FLAG (C and D), p21 CIP1 , and α-tubulin at 24 h posttransfection. (E) IFIXα1 induces p21 CIP1 mRNA expression. H1299 cells were cotransfected with p53 (0.5 μg) and 5.5 μg of FLAG-tagged empty vector (V), IFIXα1 (α1), IFIX-HIN (HIN), or IFIX-N (N). At 24 h posttransfection, total RNA (10 μg) isolated from these cells was analyzed by Northern blotting using p21 CIP1 or IFIX cDNA as a probe. The 18S and 28S rRNAs served as loading controls.

    Journal: Molecular and Cellular Biology

    Article Title: Interferon-Inducible Protein IFIX?1 Functions as a Negative Regulator of HDM2

    doi: 10.1128/MCB.26.5.1979-1996.2006

    Figure Lengend Snippet: IFIX-HIN is sufficient to downregulate HDM2. (A) IFIX-HIN downregulates HDM2 expression. 293T cells were transfected with EGFP empty vector (EGFP) or EGFP-IFIX-HIN. MG132 (10 μM) treatment started at 5 h before harvest. Cell lysates isolated from the GFP-positive cells were analyzed by Western blotting using antibodies against HDM2, EGFP, and α-tubulin. (B to D) IFIX-HIN induces p53 and p21 CIP1 . H1299 cells were transfected with p53 (0.1 μg) and increasing amounts (0.5, 1.0, and 1.8 μg) of the FLAG-tagged IFIXα1 (B), IFIX-HIN (C), or IFIX-N (D), followed by Western blotting using antibodies against p53, IFIXα (B), FLAG (C and D), p21 CIP1 , and α-tubulin at 24 h posttransfection. (E) IFIXα1 induces p21 CIP1 mRNA expression. H1299 cells were cotransfected with p53 (0.5 μg) and 5.5 μg of FLAG-tagged empty vector (V), IFIXα1 (α1), IFIX-HIN (HIN), or IFIX-N (N). At 24 h posttransfection, total RNA (10 μg) isolated from these cells was analyzed by Northern blotting using p21 CIP1 or IFIX cDNA as a probe. The 18S and 28S rRNAs served as loading controls.

    Article Snippet: To determine their ability to bind HDM2, we cotransfected 293T cells with HDM2 and EGFP, EGFP-tagged IFIXα1, IFIX-N, or IFIX-HIN, followed by IP using anti-GFP (Fig. , left panel) or anti-HDM2 (Fig. , right panel) antibody and Western blotting with either anti-HDM2 or anti-GFP antibody.

    Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Western Blot, Northern Blot

    TCTP inhibits anticancer drug-induced cell death and cleavage of EGFR and PLC-γ in HeLa cells. (A) TCTP-induced inhibition of cytotoxic drug-induced cell death. AdNull- and adTCTP-infected HeLa cells (MOI, 10) were incubated with 20 μM etoposide or 0.1 μM taxol and then DNA fragmentation was analyzed using PI staining and FACS as described in Materials and Methods. (B) TCTP-induced inhibition of cytotoxic drug-induced EGFR and PLC-γ fragmentation. After treatment of 20 μM etoposide or 0.1 μM taxol, adGFP (G)- and adTCTP-GFP (T)-infected HeLa cell (MOI, 10) extracts were blotted with anti-EGFR, -PLC- γ, and -GFP antibodies.

    Journal: BMC Cancer

    Article Title: Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells

    doi: 10.1186/1471-2407-14-165

    Figure Lengend Snippet: TCTP inhibits anticancer drug-induced cell death and cleavage of EGFR and PLC-γ in HeLa cells. (A) TCTP-induced inhibition of cytotoxic drug-induced cell death. AdNull- and adTCTP-infected HeLa cells (MOI, 10) were incubated with 20 μM etoposide or 0.1 μM taxol and then DNA fragmentation was analyzed using PI staining and FACS as described in Materials and Methods. (B) TCTP-induced inhibition of cytotoxic drug-induced EGFR and PLC-γ fragmentation. After treatment of 20 μM etoposide or 0.1 μM taxol, adGFP (G)- and adTCTP-GFP (T)-infected HeLa cell (MOI, 10) extracts were blotted with anti-EGFR, -PLC- γ, and -GFP antibodies.

    Article Snippet: Anti-EGFR, and -GFP antibodies were from Santa Cruz (Santa Cruz, CA).

    Techniques: Planar Chromatography, Inhibition, Infection, Incubation, Staining, FACS

    Par3 forms a complex with BACE1 and regulates its trafficking (a) Hippocampal neurons were transfected with indicated constructs. At DIV11 neurons were immunostained for FLAG (red) and TGN (green). Merge shows the overlap between BACE1 and TGN. Scale bar: 10μm. (b) Quantification of colocalization of BACE1 and TGN. (c) Hippocampal neurons were transfected with indicated constructs together with hBACE1-GFP and LAMP1-mRFP and imaged live on DIV11. Scale bar: 10μm. (d) Quantification of colocalization of hBACE1-GFP and LAMP1-mRFP. Data were expressed as Mean ± SEM with Student’s t test, *p

    Journal: Neurobiology of aging

    Article Title: Par3 and aPKC regulate BACE1 endosome-to-TGN trafficking through PACS1

    doi: 10.1016/j.neurobiolaging.2017.08.024

    Figure Lengend Snippet: Par3 forms a complex with BACE1 and regulates its trafficking (a) Hippocampal neurons were transfected with indicated constructs. At DIV11 neurons were immunostained for FLAG (red) and TGN (green). Merge shows the overlap between BACE1 and TGN. Scale bar: 10μm. (b) Quantification of colocalization of BACE1 and TGN. (c) Hippocampal neurons were transfected with indicated constructs together with hBACE1-GFP and LAMP1-mRFP and imaged live on DIV11. Scale bar: 10μm. (d) Quantification of colocalization of hBACE1-GFP and LAMP1-mRFP. Data were expressed as Mean ± SEM with Student’s t test, *p

    Article Snippet: For Western blot analysis, the primary antibodies used were rabbit anti-Par3 antibody (1:5000; Cat. No. 07-330, Millipore), rabbit anti-BACE1 antibody (1:2000, D10E5, Cell Signaling), rabbit anti-phospho-BACE pSer498 antibody (1:2000, PA5-12549, ThermoFisher), rabbit anti-PACS1 antibody (1:2000, a generous gift from Dr. Gary Thomas), mouse anti-GAPDH antibody (1:8000, 6C5, Millipore), mouse anti-FLAG antibody (1:2000, M2, Sigma- Aldrich) and rabbit anti-GFP antibody (1:1000, A-11122, Life-technologies).

    Techniques: Transfection, Construct

    Tissue-resident Nest-GFP(+) cells and not BM-derived MSCs contribute to remodeling of tumor blood vessels . (A) B16F10 tumor cells were subcutaneously transplanted into the flank of Nest-GFP mice. Tumors were removed 21 days after tumor induction and subjected to immunofluorescence analysis. Vessels were stained for CD31 (endothelial cells), smooth muscle actin (SMA; pericytes/SMC), and GFP or Nestin, respectively. Control sections were stained with isotype controls. Nest-Control, Nest-GFP transgenic mice. Scale bar 15 μm. (B) Immunofluorescence analysis of B16F10 tumors grown subcutaneously in the flank of BM-reconstituted Nest-GFP mice. NestwtBM, Nest-GFP mice received bone-marrow (BM) from wt (C57Bl/6) mice after lethally total body irradiation (7 + 3Gy). (C) Experiment with reciprocal BM chimera as shown in (B) . wtNestBM, wt mice received bone-marrow (BM) from Nest-GFP mice after lethally total body irradiation. Scale bar 20 μm. (D) GFP-expression was quantified by counting GFP- and SMA-immunoreactive vascular structures (pericytes/SMC-stabilized vessels). (E) GFP-expression was further analyzed using Western blot analysis.

    Journal: Frontiers in Oncology

    Article Title: Nestin(+) Tissue-Resident Multipotent Stem Cells Contribute to Tumor Progression by Differentiating into Pericytes and Smooth Muscle Cells Resulting in Blood Vessel Remodeling

    doi: 10.3389/fonc.2014.00169

    Figure Lengend Snippet: Tissue-resident Nest-GFP(+) cells and not BM-derived MSCs contribute to remodeling of tumor blood vessels . (A) B16F10 tumor cells were subcutaneously transplanted into the flank of Nest-GFP mice. Tumors were removed 21 days after tumor induction and subjected to immunofluorescence analysis. Vessels were stained for CD31 (endothelial cells), smooth muscle actin (SMA; pericytes/SMC), and GFP or Nestin, respectively. Control sections were stained with isotype controls. Nest-Control, Nest-GFP transgenic mice. Scale bar 15 μm. (B) Immunofluorescence analysis of B16F10 tumors grown subcutaneously in the flank of BM-reconstituted Nest-GFP mice. NestwtBM, Nest-GFP mice received bone-marrow (BM) from wt (C57Bl/6) mice after lethally total body irradiation (7 + 3Gy). (C) Experiment with reciprocal BM chimera as shown in (B) . wtNestBM, wt mice received bone-marrow (BM) from Nest-GFP mice after lethally total body irradiation. Scale bar 20 μm. (D) GFP-expression was quantified by counting GFP- and SMA-immunoreactive vascular structures (pericytes/SMC-stabilized vessels). (E) GFP-expression was further analyzed using Western blot analysis.

    Article Snippet: Reagents and antibodies Nestin antibody was from Acris Antibodies (Herford, Germany), alpha smooth muscle actin (SMA) and CD34 antibodies were from Santa Cruz (Santa Cruz, USA), CD31 antibody was from Dianova (Hamburg, Germany), and GFP antibody was from Thermo Scientific (Dreieich, Germany).

    Techniques: Derivative Assay, Mouse Assay, Immunofluorescence, Staining, Transgenic Assay, Irradiation, Expressing, Western Blot

    Tissue-resident Nest-GFP(+) pericytes associate with angiogenic vessels in LLC tumors grown in Nestin-GFP mice . (A) Immunofluorescence analysis of LLC tumors grown subcutaneously in the flank of wt BM-reconstituted Nest-GFP mice (NestwtBM). Tumors were isolated at day 21 and subjected for immunofluorescence analysis. Vessels were stained for CD31 or SMA and GFP. (B) Experiment with reciprocal BM chimera as shown in (A) . wtNestBM, wt mice having received BM from Nest-GFP mice after total body irradiation. To further confirm that tissue-resident MSCs and not BM-derived MSCs contribute to vascular remodeling processes one group of irradiated animals received NestBM for hematopoietic reconstitution that was supplemented with BM-derived EGFP-labeled MSC. Control sections were stained with isotype controls. Scale bar 20 μm. (C,D) GFP-expression was quantified by counting GFP and SMA-immunoreactive vessels and further analyzed using Western blot analysis.

    Journal: Frontiers in Oncology

    Article Title: Nestin(+) Tissue-Resident Multipotent Stem Cells Contribute to Tumor Progression by Differentiating into Pericytes and Smooth Muscle Cells Resulting in Blood Vessel Remodeling

    doi: 10.3389/fonc.2014.00169

    Figure Lengend Snippet: Tissue-resident Nest-GFP(+) pericytes associate with angiogenic vessels in LLC tumors grown in Nestin-GFP mice . (A) Immunofluorescence analysis of LLC tumors grown subcutaneously in the flank of wt BM-reconstituted Nest-GFP mice (NestwtBM). Tumors were isolated at day 21 and subjected for immunofluorescence analysis. Vessels were stained for CD31 or SMA and GFP. (B) Experiment with reciprocal BM chimera as shown in (A) . wtNestBM, wt mice having received BM from Nest-GFP mice after total body irradiation. To further confirm that tissue-resident MSCs and not BM-derived MSCs contribute to vascular remodeling processes one group of irradiated animals received NestBM for hematopoietic reconstitution that was supplemented with BM-derived EGFP-labeled MSC. Control sections were stained with isotype controls. Scale bar 20 μm. (C,D) GFP-expression was quantified by counting GFP and SMA-immunoreactive vessels and further analyzed using Western blot analysis.

    Article Snippet: Reagents and antibodies Nestin antibody was from Acris Antibodies (Herford, Germany), alpha smooth muscle actin (SMA) and CD34 antibodies were from Santa Cruz (Santa Cruz, USA), CD31 antibody was from Dianova (Hamburg, Germany), and GFP antibody was from Thermo Scientific (Dreieich, Germany).

    Techniques: Mouse Assay, Immunofluorescence, Isolation, Staining, Irradiation, Derivative Assay, Labeling, Expressing, Western Blot

    Transduction of Gsx2 changes the cell fate of postnatal OB progenitor cells in vivo . Retroviral particles expressing EGFP or Gsx2-EGFP were injected into the OB SEZ of P4 mice, and OB sections were obtained at P9 and immunostained with antibodies against GFP and Pax6 (A, B), Olig2 (C, D) or Dcx (E, F). Arrows and arrowheads indicate GFP + cells expressing a particular cell marker, and the insets show high magnification images of double-labeled cells indicated with arrowheads in the main images. Gsx2 overexpression caused a 59.4% reduction (*P

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Transduction of Gsx2 changes the cell fate of postnatal OB progenitor cells in vivo . Retroviral particles expressing EGFP or Gsx2-EGFP were injected into the OB SEZ of P4 mice, and OB sections were obtained at P9 and immunostained with antibodies against GFP and Pax6 (A, B), Olig2 (C, D) or Dcx (E, F). Arrows and arrowheads indicate GFP + cells expressing a particular cell marker, and the insets show high magnification images of double-labeled cells indicated with arrowheads in the main images. Gsx2 overexpression caused a 59.4% reduction (*P

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Transduction, In Vivo, Expressing, Injection, Mouse Assay, Marker, Labeling, Over Expression

    Effect of Gsx2 transduction on the proliferation of OB progenitor cells in vivo . Retroviral particles expressing EGFP or Gsx2-EGFP were injected into the OB SEZ of P4 mice. OB sections obtained at P7 were immunostained with antibodies against GFP and the mitotic marker Phospho-Histone-H3 (PH-H3) (A, B) or the cell cycle marker Ki67 (C, D). The insets show high magnification images of the double-labeled cells indicated with arrowheads in the main images. Gsx2 overexpression caused a 45% reduction in the proportion of progenitors in mitosis (E) when compared with the controls, whereas the total number of cycling cells (F) was not significantly affected. Graphs in E and F represent the mean percentage ± s.e.m. (n = 5 animals per condition). *P

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Effect of Gsx2 transduction on the proliferation of OB progenitor cells in vivo . Retroviral particles expressing EGFP or Gsx2-EGFP were injected into the OB SEZ of P4 mice. OB sections obtained at P7 were immunostained with antibodies against GFP and the mitotic marker Phospho-Histone-H3 (PH-H3) (A, B) or the cell cycle marker Ki67 (C, D). The insets show high magnification images of the double-labeled cells indicated with arrowheads in the main images. Gsx2 overexpression caused a 45% reduction in the proportion of progenitors in mitosis (E) when compared with the controls, whereas the total number of cycling cells (F) was not significantly affected. Graphs in E and F represent the mean percentage ± s.e.m. (n = 5 animals per condition). *P

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Transduction, In Vivo, Expressing, Injection, Mouse Assay, Marker, Labeling, Over Expression

    Injection of retroviral particles to express Gsx2 in dividing OB progenitor cells in vivo . Retroviral particles expressing EGFP or Gsx2-EGFP were injected into the OB subependymal zone (SEZ) of P4 mice to infect dividing progenitor cells. The mice were analyzed at P7, when GFP-labeled cells were mainly found in the SEZ. Low magnification images of OB coronal sections show the distribution of infected cells after immunostaining for GFP (A, B). Immunostaining for Gsx2 and GFP show that 69.85% of GFP + cells expressed Gsx2 in animals injected with Gsx2-EGFP particles (D, E) compared to only 0.61% in animals injected with EGFP particles (C, E). The insets in C, D are high magnification images of the cells indicated by the arrowheads in the main images. The graph in E represents the mean percentage ± s.e.m. (n = 3 animals per condition) of the infected cells expressing both Gsx2 and GFP. ***P

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Injection of retroviral particles to express Gsx2 in dividing OB progenitor cells in vivo . Retroviral particles expressing EGFP or Gsx2-EGFP were injected into the OB subependymal zone (SEZ) of P4 mice to infect dividing progenitor cells. The mice were analyzed at P7, when GFP-labeled cells were mainly found in the SEZ. Low magnification images of OB coronal sections show the distribution of infected cells after immunostaining for GFP (A, B). Immunostaining for Gsx2 and GFP show that 69.85% of GFP + cells expressed Gsx2 in animals injected with Gsx2-EGFP particles (D, E) compared to only 0.61% in animals injected with EGFP particles (C, E). The insets in C, D are high magnification images of the cells indicated by the arrowheads in the main images. The graph in E represents the mean percentage ± s.e.m. (n = 3 animals per condition) of the infected cells expressing both Gsx2 and GFP. ***P

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Injection, In Vivo, Expressing, Mouse Assay, Labeling, Infection, Immunostaining

    Transduction of Gsx2 decreases the capacity of GESCs to differentiate into neurons, astrocytes and oligodendrocytes. E13.5 GESCs transduced with viral particles expressing EGFP and Gsx2-EGFP were plated under differentiation conditions for 6 days. Dual immunostaining with GFP and TuJ1, GFAP or O4 antibodies revealed that the populations of neurons (A, B, I), astrocytes (C, D, I) and oligodendrocytes (E, F, I) were markedly reduced (3.8 to 8-fold fewer) in the Gsx2-EGFP infected cultures when compared to the controls. The proportion of infected cells labeled with an antibody against Ki67 was 9.7-fold greater in the Gsx2-EGFP infected cultures than in the controls (G–I). Arrows and arrowheads indicate GFP + cells expressing a particular cell marker, and the insets show high magnification images of the double-labeled cells indicated with arrowheads in the main images. The graph in I represents the mean percentage ± s.e.m. (n = 3 cultures per condition). **P

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Transduction of Gsx2 decreases the capacity of GESCs to differentiate into neurons, astrocytes and oligodendrocytes. E13.5 GESCs transduced with viral particles expressing EGFP and Gsx2-EGFP were plated under differentiation conditions for 6 days. Dual immunostaining with GFP and TuJ1, GFAP or O4 antibodies revealed that the populations of neurons (A, B, I), astrocytes (C, D, I) and oligodendrocytes (E, F, I) were markedly reduced (3.8 to 8-fold fewer) in the Gsx2-EGFP infected cultures when compared to the controls. The proportion of infected cells labeled with an antibody against Ki67 was 9.7-fold greater in the Gsx2-EGFP infected cultures than in the controls (G–I). Arrows and arrowheads indicate GFP + cells expressing a particular cell marker, and the insets show high magnification images of the double-labeled cells indicated with arrowheads in the main images. The graph in I represents the mean percentage ± s.e.m. (n = 3 cultures per condition). **P

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Transduction, Expressing, Immunostaining, Infection, Labeling, Marker

    Effect of Gsx2 on NSC proliferation. Infected E13.5 OBSCs, E13.5 GESCs, and P4 OBSCs were grown in proliferation conditions for 3–4 days and labeled with BrdU 20 hours prior to fixation. Immunostaining for nestin and EGFP indicated that the vast majority of cells in both infected cultures are neuroepithelial cells (A, B). Immunostaining with antibodies against BrdU and GFP revealed that the majority of GFP + cells in the controls had incorporated BrdU (C, E, G, I–K). Transduction of Gsx2 caused a 27–46% reduction in the cells labeled for BrdU + and EGFP + (D, F, H–K). The insets show high magnification images of the dual-labeled cells indicated with arrowheads in the main images. Graphs I–K represent the mean percentage (mean ± s.e.m., n = 3–6 cultures per condition). **P

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Effect of Gsx2 on NSC proliferation. Infected E13.5 OBSCs, E13.5 GESCs, and P4 OBSCs were grown in proliferation conditions for 3–4 days and labeled with BrdU 20 hours prior to fixation. Immunostaining for nestin and EGFP indicated that the vast majority of cells in both infected cultures are neuroepithelial cells (A, B). Immunostaining with antibodies against BrdU and GFP revealed that the majority of GFP + cells in the controls had incorporated BrdU (C, E, G, I–K). Transduction of Gsx2 caused a 27–46% reduction in the cells labeled for BrdU + and EGFP + (D, F, H–K). The insets show high magnification images of the dual-labeled cells indicated with arrowheads in the main images. Graphs I–K represent the mean percentage (mean ± s.e.m., n = 3–6 cultures per condition). **P

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Infection, Labeling, Immunostaining, Transduction

    Gsx2 can be efficiently transduced in neural stem cells (NSCs) using retroviral vectors. A) Schematic representation of the constructs used to express Gsx2 and EGFP in NSCs and progenitor cells, both in culture and in vivo . B–I) Cells isolated from the OB of E13.5 mice were cultured and passaged as floating neurospheres in the presence of FGF-2 and EGF, and they were then infected with the corresponding viral supernatants. Gsx2 + cells were not observed in EGFP-infected OBSC cultures growing under conditions of proliferation (B, D) or differentiation (F, H). By contrast, the majority of GFP + cells (detected by immunostaining) in the Gsx2-EGFP infected cultures expressed Gsx2 while growing under proliferation (85.24%±2.62; Prolif.: C, E, J) or differentiation conditions (for at least 20 days in the presence of 5% FBS, 83.27%±3.90, Dif.: G, I, J). The graph in J represents the mean percentage ± s.e.m. (n = 3 cultures per condition) of cells expressing both Gsx2 and GFP. Scale bar (shown in I) = 60.5 µm.

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Gsx2 can be efficiently transduced in neural stem cells (NSCs) using retroviral vectors. A) Schematic representation of the constructs used to express Gsx2 and EGFP in NSCs and progenitor cells, both in culture and in vivo . B–I) Cells isolated from the OB of E13.5 mice were cultured and passaged as floating neurospheres in the presence of FGF-2 and EGF, and they were then infected with the corresponding viral supernatants. Gsx2 + cells were not observed in EGFP-infected OBSC cultures growing under conditions of proliferation (B, D) or differentiation (F, H). By contrast, the majority of GFP + cells (detected by immunostaining) in the Gsx2-EGFP infected cultures expressed Gsx2 while growing under proliferation (85.24%±2.62; Prolif.: C, E, J) or differentiation conditions (for at least 20 days in the presence of 5% FBS, 83.27%±3.90, Dif.: G, I, J). The graph in J represents the mean percentage ± s.e.m. (n = 3 cultures per condition) of cells expressing both Gsx2 and GFP. Scale bar (shown in I) = 60.5 µm.

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Construct, In Vivo, Isolation, Mouse Assay, Cell Culture, Infection, Immunostaining, Expressing

    Transduction of Gsx2 decreases the capacity of OBSCs to differentiate into astrocytes and oligodendrocytes. E13.5 OBSCs transduced with viral particles expressing EGFP and Gsx2-EGFP were plated under differentiation conditions for 6 (not shown) and 20 days (A–F). Dual immunostaining with GFP and TuJ1, GFAP or O4 antibodies revealed the populations of neurons (A, B) and oligodendrocytes (E, F) to be similar in cultures infected with either virus. However, GFAP + -GFP + cells were 32% fewer in the Gsx2-EGFP infected cultures than in those infected with EGFP alone (C, D, G). The insets show high magnification images of the double-labeled cells indicated with arrowheads in the main images. The graph in G represents the mean percentage ± s.e.m. (n = 8 cultures per condition; data from 6 and 20 days of differentiation were combined). *P

    Journal: PLoS ONE

    Article Title: The Homeobox Gene Gsx2 Regulates the Self-Renewal and Differentiation of Neural Stem Cells and the Cell Fate of Postnatal Progenitors

    doi: 10.1371/journal.pone.0029799

    Figure Lengend Snippet: Transduction of Gsx2 decreases the capacity of OBSCs to differentiate into astrocytes and oligodendrocytes. E13.5 OBSCs transduced with viral particles expressing EGFP and Gsx2-EGFP were plated under differentiation conditions for 6 (not shown) and 20 days (A–F). Dual immunostaining with GFP and TuJ1, GFAP or O4 antibodies revealed the populations of neurons (A, B) and oligodendrocytes (E, F) to be similar in cultures infected with either virus. However, GFAP + -GFP + cells were 32% fewer in the Gsx2-EGFP infected cultures than in those infected with EGFP alone (C, D, G). The insets show high magnification images of the double-labeled cells indicated with arrowheads in the main images. The graph in G represents the mean percentage ± s.e.m. (n = 8 cultures per condition; data from 6 and 20 days of differentiation were combined). *P

    Article Snippet: Sections containing the OB were collected and immunostained with antibodies against GFP, Gsx2, Phospho-Histone H3 (PH-H3, rabbit polyclonal 1∶1,000: Upstate, Cat. n. 06-570), Ki67 (rabbit monoclonal, 1∶1,000: Thermo Sci., Fremont, CA, Cat. n. RM-9106); Olig2 (rabbit polyclonal 1∶2,000: Millipore, Cat. n. AB9610); doublecortin (Dcx, guinea pig polyclonal 1∶3,000: Millipore, Cat. n. AB2253) and Pax6 (rabbit polyclonal 1∶300: Covance, Cat. n. PRB-278P).

    Techniques: Transduction, Expressing, Immunostaining, Infection, Labeling

    Silencing calcineurin expression and a pharmacological inhibitor of calcineurin decrease 5-HT uptake via SERT. A , HEK-293 cells were transfected with the plasmid encoding SERT and control or CaNA siRNA. Total protein extracts were analyzed by Western blotting using the monoclonal anti-GFP antibody, the polyclonal anti-CaNA antibody, and the anti-GAPDH antibody. B , [ 3 H]-5-HT uptake in HEK-293 cells expressing SERT in the presence of control or CaNA siRNA. A representative experiment is shown. Data for V max of 5-HT uptake, expressed in percentage of value in cells transfected with control siRNA (3.78 ± 0.49 pmol/min), are the mean ± SEM of values measured in three independent experiments. ** p

    Journal: The Journal of Neuroscience

    Article Title: Calcineurin Interacts with the Serotonin Transporter C-Terminus to Modulate Its Plasma Membrane Expression and Serotonin Uptake

    doi: 10.1523/JNEUROSCI.0076-13.2013

    Figure Lengend Snippet: Silencing calcineurin expression and a pharmacological inhibitor of calcineurin decrease 5-HT uptake via SERT. A , HEK-293 cells were transfected with the plasmid encoding SERT and control or CaNA siRNA. Total protein extracts were analyzed by Western blotting using the monoclonal anti-GFP antibody, the polyclonal anti-CaNA antibody, and the anti-GAPDH antibody. B , [ 3 H]-5-HT uptake in HEK-293 cells expressing SERT in the presence of control or CaNA siRNA. A representative experiment is shown. Data for V max of 5-HT uptake, expressed in percentage of value in cells transfected with control siRNA (3.78 ± 0.49 pmol/min), are the mean ± SEM of values measured in three independent experiments. ** p

    Article Snippet: The mouse monoclonal anti-GFP antibody (mixture of clones 7.1 and 13.1) was from Roche Diagnostics, the rabbit polyclonal anti-GFP antibody from Invitrogen, the rabbit polyclonal anti-SERT antibody from ImmunoStar, the rabbit polyclonal anti-CaNA and CaNB antibody from Millipore Bioscience Research Reagents, the rabbit polyclonal anti-CaNA antibody from Millipore, the mouse monoclonal anti-CaNA antibody and the polyclonal anti-Flag antibody from Sigma-Aldrich, and the rabbit anti-GAPDH antibody from Santa Cruz Biotechnology.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    Structure of TCF-4-P2/3 mutants and analysis of TCF transcriptional activity after co-transfection with wild-type HIPK2 or a kinase-inactive mutant (A) Amino acid sequence of TCF-4J, TCF-4K, and TCF-4-P2/3 mutants (TCF-4J-P2/3 and TCF-4K-P2/3) showing substitution of (Ser 154 , Ser 156 , Thr 178 , and Thr 189 ) to alanine. Mutation sites are indicated by bold letters. (B-C) Expressions of TCF-4 and HIPK2 in total cell lysate (TCL) and nuclear extracts (Nuc) immunoprecipitated (IP) with anti-c-Myc antibody, and TCF transcriptional activity of TCF-4J, TCF-4K, and TCF-4-P2/3 mutants (TCF-4J-P2/3 and TCF-4K-P2/3) after co-transfection with wild-type HIPK2 (wt) or a kinase-inactive mutant (mt) in HAK-1A (B) and Huh7 (C) cells. Expression levels of TCF-4 and HIPK2 were evaluated by Western blot analysis using antibodies against Myc-tag (arrow) and GFP, respectively. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p

    Journal: Cancer letters

    Article Title: The SxxSS motif of T-cell factor-4 isoforms modulates Wnt/β-catenin signal activation in hepatocellular carcinoma cells

    doi: 10.1016/j.canlet.2013.03.031

    Figure Lengend Snippet: Structure of TCF-4-P2/3 mutants and analysis of TCF transcriptional activity after co-transfection with wild-type HIPK2 or a kinase-inactive mutant (A) Amino acid sequence of TCF-4J, TCF-4K, and TCF-4-P2/3 mutants (TCF-4J-P2/3 and TCF-4K-P2/3) showing substitution of (Ser 154 , Ser 156 , Thr 178 , and Thr 189 ) to alanine. Mutation sites are indicated by bold letters. (B-C) Expressions of TCF-4 and HIPK2 in total cell lysate (TCL) and nuclear extracts (Nuc) immunoprecipitated (IP) with anti-c-Myc antibody, and TCF transcriptional activity of TCF-4J, TCF-4K, and TCF-4-P2/3 mutants (TCF-4J-P2/3 and TCF-4K-P2/3) after co-transfection with wild-type HIPK2 (wt) or a kinase-inactive mutant (mt) in HAK-1A (B) and Huh7 (C) cells. Expression levels of TCF-4 and HIPK2 were evaluated by Western blot analysis using antibodies against Myc-tag (arrow) and GFP, respectively. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p

    Article Snippet: Western blot analysis was carried out as previously described [ ] using primary antibodies generated against Myc-tag, TCF-4, β-catenin, CREB-binding protein (CBP), transducin-like enhancer of split (TLE), GFP, HIPK2 (Cell Signaling Technology, Beverly, MA), C-terminal-binding protein (CtBP) (Millipore, Bedford, MA), lamin A/C, and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Activity Assay, Cotransfection, Mutagenesis, Sequencing, Immunoprecipitation, Expressing, Western Blot, Luciferase

    TCF transcriptional activity of TCF-4 isoforms after co-transfection with shRNA plasmids against HIPK2 (A) GFP-shRNA and HIPK2 expression in Huh7 cells transfected with shRNA plasmids against HIPK2 (shHIPK2 #1-4) and a scrambled control. Expression of shRNA was confirmed by GFP expression (left panel). Endogenous HIPK2 expression level in nuclear extracts of the transfected cells was evaluated by Western blot analysis using antibodies against HIPK2 and lamin A/C as a protein loading control. The expression level was determined by densitometry and normalized to lamin A/C (right panel). (B-C) TCF transcriptional activity of TCF-4 isoforms after co-transfection with shRNA plasmids against HIPK2 (shHIPK2 #2 and #3) in HAK-1A (B) and Huh7 (C) cells. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p

    Journal: Cancer letters

    Article Title: The SxxSS motif of T-cell factor-4 isoforms modulates Wnt/β-catenin signal activation in hepatocellular carcinoma cells

    doi: 10.1016/j.canlet.2013.03.031

    Figure Lengend Snippet: TCF transcriptional activity of TCF-4 isoforms after co-transfection with shRNA plasmids against HIPK2 (A) GFP-shRNA and HIPK2 expression in Huh7 cells transfected with shRNA plasmids against HIPK2 (shHIPK2 #1-4) and a scrambled control. Expression of shRNA was confirmed by GFP expression (left panel). Endogenous HIPK2 expression level in nuclear extracts of the transfected cells was evaluated by Western blot analysis using antibodies against HIPK2 and lamin A/C as a protein loading control. The expression level was determined by densitometry and normalized to lamin A/C (right panel). (B-C) TCF transcriptional activity of TCF-4 isoforms after co-transfection with shRNA plasmids against HIPK2 (shHIPK2 #2 and #3) in HAK-1A (B) and Huh7 (C) cells. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p

    Article Snippet: Western blot analysis was carried out as previously described [ ] using primary antibodies generated against Myc-tag, TCF-4, β-catenin, CREB-binding protein (CBP), transducin-like enhancer of split (TLE), GFP, HIPK2 (Cell Signaling Technology, Beverly, MA), C-terminal-binding protein (CtBP) (Millipore, Bedford, MA), lamin A/C, and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Activity Assay, Cotransfection, shRNA, Expressing, Transfection, Western Blot, Luciferase

    Structure of TCF-4K with mutations in the SxxSS motif and P2/3 sites and their TCF transcriptional activity after co-transfection with wild-type HIPK2 or a kinase-inactive mutant (A) Amino acid sequence of TCF-4K with mutations in SxxSS motif and P2/3 sites (269A-P2/3, 272A-P2/3, and 273A-P2/3) showing substitution of (Ser 154 , Ser 156 , Thr 178 , Thr 189 , Ser 269 , Ser 272 , and Ser 273 ) to alanine. Mutation sites are indicated by bold letters. (B-C) Expressions of TCF-4 and HIPK2 in total cell lysate, and TCF transcriptional activity of the mutants after co-transfection with wild-type HIPK2 (wt) or a kinase-inactive mutant (mt) in HAK-1A (B) and Huh7 (C) cells. Expression levels of TCF-4 and HIPK2 were evaluated by Western blot analysis using antibodies against Myc-tag (arrow) and GFP, respectively. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p

    Journal: Cancer letters

    Article Title: The SxxSS motif of T-cell factor-4 isoforms modulates Wnt/β-catenin signal activation in hepatocellular carcinoma cells

    doi: 10.1016/j.canlet.2013.03.031

    Figure Lengend Snippet: Structure of TCF-4K with mutations in the SxxSS motif and P2/3 sites and their TCF transcriptional activity after co-transfection with wild-type HIPK2 or a kinase-inactive mutant (A) Amino acid sequence of TCF-4K with mutations in SxxSS motif and P2/3 sites (269A-P2/3, 272A-P2/3, and 273A-P2/3) showing substitution of (Ser 154 , Ser 156 , Thr 178 , Thr 189 , Ser 269 , Ser 272 , and Ser 273 ) to alanine. Mutation sites are indicated by bold letters. (B-C) Expressions of TCF-4 and HIPK2 in total cell lysate, and TCF transcriptional activity of the mutants after co-transfection with wild-type HIPK2 (wt) or a kinase-inactive mutant (mt) in HAK-1A (B) and Huh7 (C) cells. Expression levels of TCF-4 and HIPK2 were evaluated by Western blot analysis using antibodies against Myc-tag (arrow) and GFP, respectively. TCF transcriptional activity was calculated based on luciferase and β-galactosidase activity. The results are expressed as the mean ± SD. *, p

    Article Snippet: Western blot analysis was carried out as previously described [ ] using primary antibodies generated against Myc-tag, TCF-4, β-catenin, CREB-binding protein (CBP), transducin-like enhancer of split (TLE), GFP, HIPK2 (Cell Signaling Technology, Beverly, MA), C-terminal-binding protein (CtBP) (Millipore, Bedford, MA), lamin A/C, and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Activity Assay, Cotransfection, Mutagenesis, Sequencing, Expressing, Western Blot, Luciferase

    Comparative expression of differentiation markers by 18IM, MR cells, and REFs. ( A ) Immunostainings on primary rat embryonic fibroblasts (REFs) and GFP-S18–2 transformed cells (18IM). Green signal, marker expression. Blue signal, DNA (stained with

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MRPS18-2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties

    doi: 10.1073/pnas.0911545106

    Figure Lengend Snippet: Comparative expression of differentiation markers by 18IM, MR cells, and REFs. ( A ) Immunostainings on primary rat embryonic fibroblasts (REFs) and GFP-S18–2 transformed cells (18IM). Green signal, marker expression. Blue signal, DNA (stained with

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-beta-III isoform of tubulin (Millipore); anti-keratin, Pan Ab-1 (Thermo Scientific); anti-rat MHC class II (kind gift of Helena Erlandsson-Harris, CMM, Karolinska Hospital, Stockholm); anti-smooth muscle actin (DAKO); anti- stage specific embryonic antigen SSEA-1, detecting lactoseries oligosaccharide antigen that is expressed on the surface of mouse embryonal carcinoma and embryonic stem cells, and anti-SSEA-4, (R & D Systems); anti-vimentin (DAKO), anti-GFP (Cell Signaling Technology); anti-S18–2 [clone 75–5 ( )] antibodies; Oil Red-O (Sigma–Aldrich).

    Techniques: Expressing, Transformation Assay, Marker, Staining

    Immunostaining of the tumors produced by GFP-S18–2 transformed cells in SCID mice. Formalin fixed paraffin embedded tissue was sectioned (5-μm). Paraffin was removed by xylene, followed by ethanol. Epitopes were recovered by boiling in

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MRPS18-2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties

    doi: 10.1073/pnas.0911545106

    Figure Lengend Snippet: Immunostaining of the tumors produced by GFP-S18–2 transformed cells in SCID mice. Formalin fixed paraffin embedded tissue was sectioned (5-μm). Paraffin was removed by xylene, followed by ethanol. Epitopes were recovered by boiling in

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-beta-III isoform of tubulin (Millipore); anti-keratin, Pan Ab-1 (Thermo Scientific); anti-rat MHC class II (kind gift of Helena Erlandsson-Harris, CMM, Karolinska Hospital, Stockholm); anti-smooth muscle actin (DAKO); anti- stage specific embryonic antigen SSEA-1, detecting lactoseries oligosaccharide antigen that is expressed on the surface of mouse embryonal carcinoma and embryonic stem cells, and anti-SSEA-4, (R & D Systems); anti-vimentin (DAKO), anti-GFP (Cell Signaling Technology); anti-S18–2 [clone 75–5 ( )] antibodies; Oil Red-O (Sigma–Aldrich).

    Techniques: Immunostaining, Produced, Transformation Assay, Mouse Assay, Formalin-fixed Paraffin-Embedded

    Lead triggers phase separation of TDP-43 and decreases its solubility. A, Purified TDP-43 LLPS in vitro is facilitated by lead (II) acetate trihydrate (Pb) in a dose-dependent manner. Representative 63× DIC images. Arrows: examples of single TDP-43 droplets. Asterisk: examples of amorphous TDP-43 consolidates. Arrowheads: high-contrast, inert 1 micron polystyrene microspheres added to aid sample focusing. Scale bar = 10 µm. B, LLPS was quantified using an ImageJ algorithm as percentage of ROI covered by droplets. Points at mean, with error bars at SEM, were fit by nonlinear regression analysis (line) and the LogEC50 calculated as 160 µM (dotted line). Induced PC12 cells accumulate insoluble TDP-43:: GFP upon treatment with lead (Pb). Immunoblots of RIPA insoluble (C), RIPA soluble (D) and total RIPA lysate (E) were probed with anti-GFP antibodies ( N = 3). F, Densitometric analysis of total TDP-43:: GFP/Actin bands (from panel E) show an increase in TDP-43:: GFP in response to 0.174 and 0.521 µM lead but an decrease in response to 4.69 µM lead. G, At 1.56 and 4.69 µM concentrations, lead significantly increases the ratio of insoluble TDP-43:: GFP to soluble TDP-43:: GFP. N = 3; mean ± SEM; ANOVA w/Dunnett’s multiple comparison test, * p

    Journal: Toxicological Sciences

    Article Title: Heavy Metal Neurotoxicants Induce ALS-Linked TDP-43 Pathology

    doi: 10.1093/toxsci/kfy267

    Figure Lengend Snippet: Lead triggers phase separation of TDP-43 and decreases its solubility. A, Purified TDP-43 LLPS in vitro is facilitated by lead (II) acetate trihydrate (Pb) in a dose-dependent manner. Representative 63× DIC images. Arrows: examples of single TDP-43 droplets. Asterisk: examples of amorphous TDP-43 consolidates. Arrowheads: high-contrast, inert 1 micron polystyrene microspheres added to aid sample focusing. Scale bar = 10 µm. B, LLPS was quantified using an ImageJ algorithm as percentage of ROI covered by droplets. Points at mean, with error bars at SEM, were fit by nonlinear regression analysis (line) and the LogEC50 calculated as 160 µM (dotted line). Induced PC12 cells accumulate insoluble TDP-43:: GFP upon treatment with lead (Pb). Immunoblots of RIPA insoluble (C), RIPA soluble (D) and total RIPA lysate (E) were probed with anti-GFP antibodies ( N = 3). F, Densitometric analysis of total TDP-43:: GFP/Actin bands (from panel E) show an increase in TDP-43:: GFP in response to 0.174 and 0.521 µM lead but an decrease in response to 4.69 µM lead. G, At 1.56 and 4.69 µM concentrations, lead significantly increases the ratio of insoluble TDP-43:: GFP to soluble TDP-43:: GFP. N = 3; mean ± SEM; ANOVA w/Dunnett’s multiple comparison test, * p

    Article Snippet: Blots were incubated with the following antibodies: 1:1000 anti-phospho-S409/410 TDP-43 (a gift from Leonard Petrucelli, Mayo Clinic, Rb3655), 1:2000 anti-GFP (Sigma; G1544), 1:2000 anti-TDP-43 (ProteinTech; 12892–1-AP); and 1:10000 anti-Actin (Millipore; MAB1501).

    Techniques: Solubility, Purification, In Vitro, Western Blot

    Telomere fusions in Z4 and HeT-A mutants. (A) Mitotic chromosome preparations of third instar larvae. Z4 7.1 /Z4 7.1 , Z4 2.1 /+ and pzg 66 /+ alleles show telomere fusions. woc 964/ woc B111 mutants were used as a positive control and w 1118 as negative control. Red arrowheads indicate telomere fusions. (B) Anaphase preparations from third instar larvae neuroblasts of the Z4 7.1 /Z4 7.1 , woc 964/ woc B111 alleles and w 1118 . Defective anaphases due to telomere fusions visible in Z4 7.1 /Z4 7.1 , woc 964/ woc B111 (positive control). Normal anaphases in w 1118 (negative control). (C) Mitotic S2 cells stained with DAPI after Z4 , HeT-A gag gene, cav (HOAP), and unspecific ( Sart1 ) RNAi treatment. Telomere fusions are observed in Z4 and HeT-A Gag RNAi mutants; RNAi for the cav and the Sart1 non-LTR retrotransposon from Bombyx mori were used as positive and negative control respectively. Red arrowheads indicate telomere fusions. (D) Percentage of telomere fusions found in mitotic S2 cells after RNAi treatment. (E) Mitotic chromosome preparations of HOAP-GFP/HOAP-GFP and Z4 7.1 /HOAP-GFP third instar larvae neuroblasts. Chromosomes stained with DAPI (blue) and HOAP-GFP fusion protein (green). HOAP is present in Z4 telomere fusions (red arrowheads). (F) Percentage of telomere fusions found in Z4 7.1 /ligIV and Z4 / JIL-1 double mutants. ligIV and JIL-1 mutants do not affect the number of telomere fusions in Z4 mutants. For each mutant a minimum of 100 metaphases/anaphases of three different preparations were analyzed.

    Journal: PLoS Genetics

    Article Title: The Chromosomal Proteins JIL-1 and Z4/Putzig Regulate the Telomeric Chromatin in Drosophila melanogaster

    doi: 10.1371/journal.pgen.1003153

    Figure Lengend Snippet: Telomere fusions in Z4 and HeT-A mutants. (A) Mitotic chromosome preparations of third instar larvae. Z4 7.1 /Z4 7.1 , Z4 2.1 /+ and pzg 66 /+ alleles show telomere fusions. woc 964/ woc B111 mutants were used as a positive control and w 1118 as negative control. Red arrowheads indicate telomere fusions. (B) Anaphase preparations from third instar larvae neuroblasts of the Z4 7.1 /Z4 7.1 , woc 964/ woc B111 alleles and w 1118 . Defective anaphases due to telomere fusions visible in Z4 7.1 /Z4 7.1 , woc 964/ woc B111 (positive control). Normal anaphases in w 1118 (negative control). (C) Mitotic S2 cells stained with DAPI after Z4 , HeT-A gag gene, cav (HOAP), and unspecific ( Sart1 ) RNAi treatment. Telomere fusions are observed in Z4 and HeT-A Gag RNAi mutants; RNAi for the cav and the Sart1 non-LTR retrotransposon from Bombyx mori were used as positive and negative control respectively. Red arrowheads indicate telomere fusions. (D) Percentage of telomere fusions found in mitotic S2 cells after RNAi treatment. (E) Mitotic chromosome preparations of HOAP-GFP/HOAP-GFP and Z4 7.1 /HOAP-GFP third instar larvae neuroblasts. Chromosomes stained with DAPI (blue) and HOAP-GFP fusion protein (green). HOAP is present in Z4 telomere fusions (red arrowheads). (F) Percentage of telomere fusions found in Z4 7.1 /ligIV and Z4 / JIL-1 double mutants. ligIV and JIL-1 mutants do not affect the number of telomere fusions in Z4 mutants. For each mutant a minimum of 100 metaphases/anaphases of three different preparations were analyzed.

    Article Snippet: Anti-GFP (Invitrogen, A11120), anti-Z4 , anti-JIL-1 (mouse, gift from Kristen Johansen), and control mouse IgG (Santa Cruz Biotechnology, sc-2025) were used for protein immunoprecipitation, and anti-HeT-A Gag, anti-Z4 and anti-JIL-1 were used in Western Blot experiments.

    Techniques: Positive Control, Negative Control, Staining, Mutagenesis

    Z4 interacts with JIL-1 and HeT-A GAG. (A) Z4 and JIL-1 immunoprecipitation was performed in S2 cells using anti-JIL-1 and anti-Z4 antibodies. Negative control experiments were performed by immunoprecipitating with unspecific IgGs. (B) Z4 and HeT-A GAG immunoprecipitation was done by transfecting S2 cells with HeT-A Gag-GFP and immunoprecipitating with αnti-GFP and αnti-Z4. Control experiments were performed by transfecting an empty GFP vector (pPL17). Presence of the recombinant protein is indicated on the top of the panel (+ and − symbols). Antibodies used for immunoprecipitation are indicated on the top. All extracts were fractionated by SDS-PAGE, western blotted, and developed with specific antibodies (indicated on the right of each figure). Molecular markers (kDa) are indicated on the left.

    Journal: PLoS Genetics

    Article Title: The Chromosomal Proteins JIL-1 and Z4/Putzig Regulate the Telomeric Chromatin in Drosophila melanogaster

    doi: 10.1371/journal.pgen.1003153

    Figure Lengend Snippet: Z4 interacts with JIL-1 and HeT-A GAG. (A) Z4 and JIL-1 immunoprecipitation was performed in S2 cells using anti-JIL-1 and anti-Z4 antibodies. Negative control experiments were performed by immunoprecipitating with unspecific IgGs. (B) Z4 and HeT-A GAG immunoprecipitation was done by transfecting S2 cells with HeT-A Gag-GFP and immunoprecipitating with αnti-GFP and αnti-Z4. Control experiments were performed by transfecting an empty GFP vector (pPL17). Presence of the recombinant protein is indicated on the top of the panel (+ and − symbols). Antibodies used for immunoprecipitation are indicated on the top. All extracts were fractionated by SDS-PAGE, western blotted, and developed with specific antibodies (indicated on the right of each figure). Molecular markers (kDa) are indicated on the left.

    Article Snippet: Anti-GFP (Invitrogen, A11120), anti-Z4 , anti-JIL-1 (mouse, gift from Kristen Johansen), and control mouse IgG (Santa Cruz Biotechnology, sc-2025) were used for protein immunoprecipitation, and anti-HeT-A Gag, anti-Z4 and anti-JIL-1 were used in Western Blot experiments.

    Techniques: Immunoprecipitation, Negative Control, Plasmid Preparation, Recombinant, SDS Page, Western Blot

    Generation of primary and secondary cardiospheres and their characteristics . ( a ) Timeline of primary CS, SDC, and secondary CS generation. Within 48 hours, secondary CSs were generated from SDCs. ( b ) Phase-contrast bright field images and step-by-step average cell numbers ( n = 5). Bar: 500 µm. ( c ) The gene expressions of Oct4 and c-kit were measured by real-time PCR. ( d ) The protein expressions of Oct4 and c-kit were measured by western blots and quantified by densitometry. ( e ) Primary and secondary CSs were positive for alkaline phosphatase (ALP) staining, but SDCs were not. The protein expressions of Oct4 and c-kit were assessed by confocal imaging. Oct4 expression was verified using Oct4 promoter-driven GFP cells. Nuclear colocalization of Oct4 was also confirmed in the single, dissociated secondary CS cells ( Supplementary Figure S1 ). Secondary CSs expressed Oct4 and c-kit more homogeneously and densely than primary CSs, whereas SDCs lacked expressions. TO-PRO-3 (nuclei). Bar: 50 µm. AU, arbitrary unit; CS, cardiosphere; FN, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; PDL, poly-D-lysine; SDC, sphere-derived cell.

    Journal: Molecular Therapy

    Article Title: Secondary Sphere Formation Enhances the Functionality of Cardiac Progenitor Cells

    doi: 10.1038/mt.2012.109

    Figure Lengend Snippet: Generation of primary and secondary cardiospheres and their characteristics . ( a ) Timeline of primary CS, SDC, and secondary CS generation. Within 48 hours, secondary CSs were generated from SDCs. ( b ) Phase-contrast bright field images and step-by-step average cell numbers ( n = 5). Bar: 500 µm. ( c ) The gene expressions of Oct4 and c-kit were measured by real-time PCR. ( d ) The protein expressions of Oct4 and c-kit were measured by western blots and quantified by densitometry. ( e ) Primary and secondary CSs were positive for alkaline phosphatase (ALP) staining, but SDCs were not. The protein expressions of Oct4 and c-kit were assessed by confocal imaging. Oct4 expression was verified using Oct4 promoter-driven GFP cells. Nuclear colocalization of Oct4 was also confirmed in the single, dissociated secondary CS cells ( Supplementary Figure S1 ). Secondary CSs expressed Oct4 and c-kit more homogeneously and densely than primary CSs, whereas SDCs lacked expressions. TO-PRO-3 (nuclei). Bar: 50 µm. AU, arbitrary unit; CS, cardiosphere; FN, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; PDL, poly-D-lysine; SDC, sphere-derived cell.

    Article Snippet: Immunofluorescent staining was performed as previously described with minor modifications., Cells or tissue sections were incubated with antibodies against GFP (Abcam, Cambridge, UK), Oct4 (Santa Cruz Biotechnology, BD), c-kit (BD), Connexin 43 (Santa Cruz Biotechnology), Troponin T (Santa Cruz Biotechnology), and Nkx2.5 (Abcam).

    Techniques: Generated, Real-time Polymerase Chain Reaction, Western Blot, ALP Assay, Staining, Imaging, Expressing, Derivative Assay

    Synaptic transmission regulates the size and density of GFP-GLT-1 clusters

    Journal: Glia

    Article Title: NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    doi: 10.1002/glia.21249

    Figure Lengend Snippet: Synaptic transmission regulates the size and density of GFP-GLT-1 clusters

    Article Snippet: Tissues were rinsed in 0.01 M PBS, extracted overnight with 1% Triton-X, blocked sequentially in 50 mM NH4 Cl and 20% horse serum (30 min each), rinsed with PBS containing 1% horse serum (PBS+HS), and incubated for 10-16 hr (4°C) in primary antibodies at the indicated dilutions: anti-GFAP mouse monoclonal (Sigma, catalog #G3893), 1:500; anti-GLT-1 rabbit polyclonal , 1:500; anti-GLT-1 guinea pig polyclonal (Chemicon, AB1783), 1:10,000; anti-synapsin-1 rabbit polyclonal (Invitrogen), 1:200; anti-GFP mouse monoclonal (Sigma, G6539), 1:500.

    Techniques: Transmission Assay

    GFP-GLT-1 in transfected astrocytes in slice cultures is concentrated in clusters within lamellae, spine-like, and filopodia-like processes

    Journal: Glia

    Article Title: NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    doi: 10.1002/glia.21249

    Figure Lengend Snippet: GFP-GLT-1 in transfected astrocytes in slice cultures is concentrated in clusters within lamellae, spine-like, and filopodia-like processes

    Article Snippet: Tissues were rinsed in 0.01 M PBS, extracted overnight with 1% Triton-X, blocked sequentially in 50 mM NH4 Cl and 20% horse serum (30 min each), rinsed with PBS containing 1% horse serum (PBS+HS), and incubated for 10-16 hr (4°C) in primary antibodies at the indicated dilutions: anti-GFAP mouse monoclonal (Sigma, catalog #G3893), 1:500; anti-GLT-1 rabbit polyclonal , 1:500; anti-GLT-1 guinea pig polyclonal (Chemicon, AB1783), 1:10,000; anti-synapsin-1 rabbit polyclonal (Invitrogen), 1:200; anti-GFP mouse monoclonal (Sigma, G6539), 1:500.

    Techniques: Transfection

    Time-lapse imaging shows remodeling of GFP-GLT-1 clusters in developing astrocytes in situ

    Journal: Glia

    Article Title: NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    doi: 10.1002/glia.21249

    Figure Lengend Snippet: Time-lapse imaging shows remodeling of GFP-GLT-1 clusters in developing astrocytes in situ

    Article Snippet: Tissues were rinsed in 0.01 M PBS, extracted overnight with 1% Triton-X, blocked sequentially in 50 mM NH4 Cl and 20% horse serum (30 min each), rinsed with PBS containing 1% horse serum (PBS+HS), and incubated for 10-16 hr (4°C) in primary antibodies at the indicated dilutions: anti-GFAP mouse monoclonal (Sigma, catalog #G3893), 1:500; anti-GLT-1 rabbit polyclonal , 1:500; anti-GLT-1 guinea pig polyclonal (Chemicon, AB1783), 1:10,000; anti-synapsin-1 rabbit polyclonal (Invitrogen), 1:200; anti-GFP mouse monoclonal (Sigma, G6539), 1:500.

    Techniques: Imaging, In Situ

    GFP-GLT-1 clusters often adjoin synapsin-1-positive presynaptic terminals

    Journal: Glia

    Article Title: NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    doi: 10.1002/glia.21249

    Figure Lengend Snippet: GFP-GLT-1 clusters often adjoin synapsin-1-positive presynaptic terminals

    Article Snippet: Tissues were rinsed in 0.01 M PBS, extracted overnight with 1% Triton-X, blocked sequentially in 50 mM NH4 Cl and 20% horse serum (30 min each), rinsed with PBS containing 1% horse serum (PBS+HS), and incubated for 10-16 hr (4°C) in primary antibodies at the indicated dilutions: anti-GFAP mouse monoclonal (Sigma, catalog #G3893), 1:500; anti-GLT-1 rabbit polyclonal , 1:500; anti-GLT-1 guinea pig polyclonal (Chemicon, AB1783), 1:10,000; anti-synapsin-1 rabbit polyclonal (Invitrogen), 1:200; anti-GFP mouse monoclonal (Sigma, G6539), 1:500.

    Techniques:

    Bilirubin activates sensory neurons in an MRGPR-dependent manner. ( A–D ) Confocal microscopy immunofluorescence images of adult mouse tissue sections from Mrgpra1 GFP animals with GFP expression under the control of the endogenous Mrgpra1 locus. ( A ) Mrgpra1 expression in dorsal root ganglia. Green depicts Mrgpra1 GFP . Red depicts anti-PLAP antibody staining where PLAP expression is controlled by the endogenous Mrgprd locus ( Mrgprd PLAP ). Blue depicts antibody staining against calcitonin gene-related peptide (CGRP). Scale bar is 50 µM. ( B ) Trigeminal ganglia (TG) stained with Mrgpra1 GFP (green) and anti-Substance P antibody (red). Scale bar is 50 μm. ( C ) Back skin stained with anti-GFP antibody (green) to visualize Mrgpra1 GFP nerve fibers in the dermis. Blue represents DAPI counterstain. Scale bar is 50 μm. ( D ) Spinal cord (SC) (lamina 1 and 2) stained with anti-GFP and IB4-564. Mrgpra1 GFP (green) staining was found in lamina two along with IB4 (red) positive terminals. Scale bar is 100 μm. ( E ) Representative whole-cell current-clamp recording of either WT or A1 KO DRG neurons. In WT DRG, bilirubin elicited action potentials in 5 out of 50 small-diameter neurons. In A1 KO DRG, bilirubin elicited action potentials in 0 out of 60 small-diameter neurons. Fisher’s exact test p

    Journal: eLife

    Article Title: Identification of a bilirubin receptor that may mediate a component of cholestatic itch

    doi: 10.7554/eLife.44116

    Figure Lengend Snippet: Bilirubin activates sensory neurons in an MRGPR-dependent manner. ( A–D ) Confocal microscopy immunofluorescence images of adult mouse tissue sections from Mrgpra1 GFP animals with GFP expression under the control of the endogenous Mrgpra1 locus. ( A ) Mrgpra1 expression in dorsal root ganglia. Green depicts Mrgpra1 GFP . Red depicts anti-PLAP antibody staining where PLAP expression is controlled by the endogenous Mrgprd locus ( Mrgprd PLAP ). Blue depicts antibody staining against calcitonin gene-related peptide (CGRP). Scale bar is 50 µM. ( B ) Trigeminal ganglia (TG) stained with Mrgpra1 GFP (green) and anti-Substance P antibody (red). Scale bar is 50 μm. ( C ) Back skin stained with anti-GFP antibody (green) to visualize Mrgpra1 GFP nerve fibers in the dermis. Blue represents DAPI counterstain. Scale bar is 50 μm. ( D ) Spinal cord (SC) (lamina 1 and 2) stained with anti-GFP and IB4-564. Mrgpra1 GFP (green) staining was found in lamina two along with IB4 (red) positive terminals. Scale bar is 100 μm. ( E ) Representative whole-cell current-clamp recording of either WT or A1 KO DRG neurons. In WT DRG, bilirubin elicited action potentials in 5 out of 50 small-diameter neurons. In A1 KO DRG, bilirubin elicited action potentials in 0 out of 60 small-diameter neurons. Fisher’s exact test p

    Article Snippet: For primary antibodies, we used rabbit antibody to CGRP (T-4239, Peninsula, 1:1,000), rabbit antibody to GFP (A-11122, Molecular Probes, 1:1,000), and Substance P (rat monoclonal from Abcam, 1:250 dilution, M09205).

    Techniques: Confocal Microscopy, Immunofluorescence, Expressing, Staining