rabbit polyclonal anti mic60  (Thermo Fisher)


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    Structured Review

    Thermo Fisher rabbit polyclonal anti mic60
    Submitochondrial localization of TMEM70 by fluorescence microscopy. A . 143B-rho + cells expressing TMEM70 cDNA were fixed and immunostained with antibodies against <t>Mitofilin/Mic60</t> (upper panel), or TMEM70 (lower panel). Confocal (left) and STED super-resolution (right) images are shown with insets magnified on the right panels. B . 143B-rho + cells expressing TMEM70-FLAG were fix ed, immunostained with antibodies against Mitofilin/Mic60 (red) and FLAG (green), counter-stained with DAPI (blue), and subjected to expansion microscopy. A representative image is shown (left panel) with an inset magnified on the right panel in separate channels (upper right) and overlay (lower right). C . Cultured human skin fibroblasts were fixed, immunostained with antibodies against TMEM70 (green), and subjected to expansion microscopy. A representative image is shown on the left and the inset magnified on the top right corner. D . Scheme representing the relative localization of Mic60 (in red) and TMEM70 (in green) in the inner membrane sub-compartments. OM: outer membrane; IBM: inner boundary membrane; ICM: inner cristae membrane. Scale bars: 5 µm full size images, 500 nm zoomed images (D, 1μm).
    Rabbit Polyclonal Anti Mic60, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "TMEM70 promotes ATP synthase assembly within cristae via transient interactions with subunit c"

    Article Title: TMEM70 promotes ATP synthase assembly within cristae via transient interactions with subunit c

    Journal: bioRxiv

    doi: 10.1101/2020.04.03.023861

    Submitochondrial localization of TMEM70 by fluorescence microscopy. A . 143B-rho + cells expressing TMEM70 cDNA were fixed and immunostained with antibodies against Mitofilin/Mic60 (upper panel), or TMEM70 (lower panel). Confocal (left) and STED super-resolution (right) images are shown with insets magnified on the right panels. B . 143B-rho + cells expressing TMEM70-FLAG were fix ed, immunostained with antibodies against Mitofilin/Mic60 (red) and FLAG (green), counter-stained with DAPI (blue), and subjected to expansion microscopy. A representative image is shown (left panel) with an inset magnified on the right panel in separate channels (upper right) and overlay (lower right). C . Cultured human skin fibroblasts were fixed, immunostained with antibodies against TMEM70 (green), and subjected to expansion microscopy. A representative image is shown on the left and the inset magnified on the top right corner. D . Scheme representing the relative localization of Mic60 (in red) and TMEM70 (in green) in the inner membrane sub-compartments. OM: outer membrane; IBM: inner boundary membrane; ICM: inner cristae membrane. Scale bars: 5 µm full size images, 500 nm zoomed images (D, 1μm).
    Figure Legend Snippet: Submitochondrial localization of TMEM70 by fluorescence microscopy. A . 143B-rho + cells expressing TMEM70 cDNA were fixed and immunostained with antibodies against Mitofilin/Mic60 (upper panel), or TMEM70 (lower panel). Confocal (left) and STED super-resolution (right) images are shown with insets magnified on the right panels. B . 143B-rho + cells expressing TMEM70-FLAG were fix ed, immunostained with antibodies against Mitofilin/Mic60 (red) and FLAG (green), counter-stained with DAPI (blue), and subjected to expansion microscopy. A representative image is shown (left panel) with an inset magnified on the right panel in separate channels (upper right) and overlay (lower right). C . Cultured human skin fibroblasts were fixed, immunostained with antibodies against TMEM70 (green), and subjected to expansion microscopy. A representative image is shown on the left and the inset magnified on the top right corner. D . Scheme representing the relative localization of Mic60 (in red) and TMEM70 (in green) in the inner membrane sub-compartments. OM: outer membrane; IBM: inner boundary membrane; ICM: inner cristae membrane. Scale bars: 5 µm full size images, 500 nm zoomed images (D, 1μm).

    Techniques Used: Fluorescence, Microscopy, Expressing, Staining, Cell Culture

    2) Product Images from "USP9X deubiquitinase couples the pluripotency network and cell metabolism to regulate ESC differentiation potential"

    Article Title: USP9X deubiquitinase couples the pluripotency network and cell metabolism to regulate ESC differentiation potential

    Journal: bioRxiv

    doi: 10.1101/2020.01.13.904904

    Characterization of USP9X E2/Y ESC. ( A ) qRT-PCR analysis of Usp9X expression in USP9X E2/Y and control ESC cultured in serum + LIF media (n = 3). Usp9X expression is normalized to GAPDH expression with value set up to 1 in USP9X +/y ESC. Primer pairs are as follow: 1, overlapping exon junction 5-6; 2, overlapping exon junction 9-10; 3, overlapping exon junction 17-18; 4, overlapping exon junction 2-3; and 5, overlapping exon junction 3-4. ( B ) Immunofluorescence analysis of USP9X in USP9X E2/Y and control ESC. USP9X was stained by indirect immunofluorescence using an Alexa-FLUOR Plus 555-secondary antibody (in red). DNA is labelled with DAPI (in blue). Relative quantification of USP9X signal intensity is provided (i.e. the x-axis represents the horizontal distance through the images and the y-axis the vertically averaged pixel intensity). Scale bar: 2 μm. ( C ) Flow cytometry analysis of USP9X levels in USP9X E2/Y and control ESC cultured in serum + LIF media. Representative experiment and USP9X amount are shown. ( D ) RT-PCR analysis of pluripotency (Dppa3, Sall4, Nanog, Oct4 and Klf4), endoderm (Sox9, FoxA2, Dab2, Pdx1 and FoxA1), mesoderm (Zeb2, Tbx5, Snai2, Nog and Eomes) and ectoderm (Pax6, Otx2, Sox1, Fgf5 and Fgf4) markers in Usp9X E2/Y and control ESC maintained in LIF + serum medium. ( E ) Kinetics of USP9X, OCT4, NANOG, SALL4, GATA4 and TUBBA abundance during differentiation of USP9X +/y ESC into embryonic bodies. Differentiation was induced by removal of LIF at day 0 and addition of retinoic acid (5 µM) at day 4 and 6. As anticipated, pluripotency factors OCT4, NANOG and SALL4 are downregulated during differentiation and GATA4 abundance rises starting on day 4. ( F ) Kinetics of USP9X abundance during differentiation of USP9X +/y ESC into XEN cells. As anticipated, pluripotency factors SALL4 is downregulated during differentiation and GATA4 abundance rises starting on day 3. ( G ) Representative extra-embryonic cells (XEN) derived from USP9X E2/Y and control ESC seen at day 6 of differentiation. RT-PCR analysis of pluripotency (Oct4 and Klf4) and differentiation (Gata6, Gata4, FoxA1, FoxA2, Sox17, Sox7 and Cldn) markers in USP9X E2/Y and control ESC at different days after induced differentiation (same protocol as in panel 1F).
    Figure Legend Snippet: Characterization of USP9X E2/Y ESC. ( A ) qRT-PCR analysis of Usp9X expression in USP9X E2/Y and control ESC cultured in serum + LIF media (n = 3). Usp9X expression is normalized to GAPDH expression with value set up to 1 in USP9X +/y ESC. Primer pairs are as follow: 1, overlapping exon junction 5-6; 2, overlapping exon junction 9-10; 3, overlapping exon junction 17-18; 4, overlapping exon junction 2-3; and 5, overlapping exon junction 3-4. ( B ) Immunofluorescence analysis of USP9X in USP9X E2/Y and control ESC. USP9X was stained by indirect immunofluorescence using an Alexa-FLUOR Plus 555-secondary antibody (in red). DNA is labelled with DAPI (in blue). Relative quantification of USP9X signal intensity is provided (i.e. the x-axis represents the horizontal distance through the images and the y-axis the vertically averaged pixel intensity). Scale bar: 2 μm. ( C ) Flow cytometry analysis of USP9X levels in USP9X E2/Y and control ESC cultured in serum + LIF media. Representative experiment and USP9X amount are shown. ( D ) RT-PCR analysis of pluripotency (Dppa3, Sall4, Nanog, Oct4 and Klf4), endoderm (Sox9, FoxA2, Dab2, Pdx1 and FoxA1), mesoderm (Zeb2, Tbx5, Snai2, Nog and Eomes) and ectoderm (Pax6, Otx2, Sox1, Fgf5 and Fgf4) markers in Usp9X E2/Y and control ESC maintained in LIF + serum medium. ( E ) Kinetics of USP9X, OCT4, NANOG, SALL4, GATA4 and TUBBA abundance during differentiation of USP9X +/y ESC into embryonic bodies. Differentiation was induced by removal of LIF at day 0 and addition of retinoic acid (5 µM) at day 4 and 6. As anticipated, pluripotency factors OCT4, NANOG and SALL4 are downregulated during differentiation and GATA4 abundance rises starting on day 4. ( F ) Kinetics of USP9X abundance during differentiation of USP9X +/y ESC into XEN cells. As anticipated, pluripotency factors SALL4 is downregulated during differentiation and GATA4 abundance rises starting on day 3. ( G ) Representative extra-embryonic cells (XEN) derived from USP9X E2/Y and control ESC seen at day 6 of differentiation. RT-PCR analysis of pluripotency (Oct4 and Klf4) and differentiation (Gata6, Gata4, FoxA1, FoxA2, Sox17, Sox7 and Cldn) markers in USP9X E2/Y and control ESC at different days after induced differentiation (same protocol as in panel 1F).

    Techniques Used: Quantitative RT-PCR, Expressing, Cell Culture, Immunofluorescence, Staining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    3) Product Images from "Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility"

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    Journal: Scientific Reports

    doi: 10.1038/srep33647

    LC3-immunolocalization in fresh and incubated sperm cells. Localization of LC3 in sperm cells was performed as described in materials and methods section (indirect immunofluorescence) with anti-LC3 antibody (1/250). LC3 protein was visualized in green. Figures ( a , b) (spermatozoa from fresh samples) ( c–e) (spermatozoa after 2 hours of incubation) ( f ) (spermatozoa after 2 hours of incubation with bafilomycin A1) are representative areas digitally augmented showing the localization of LC3. Figure ( g ) shows a representative area digitally augmented from merged images of LC3 and cells stained with MitoTracker Red CMXRos. Places where both LC3 and mitochondria colocalize are observed in yellow.
    Figure Legend Snippet: LC3-immunolocalization in fresh and incubated sperm cells. Localization of LC3 in sperm cells was performed as described in materials and methods section (indirect immunofluorescence) with anti-LC3 antibody (1/250). LC3 protein was visualized in green. Figures ( a , b) (spermatozoa from fresh samples) ( c–e) (spermatozoa after 2 hours of incubation) ( f ) (spermatozoa after 2 hours of incubation with bafilomycin A1) are representative areas digitally augmented showing the localization of LC3. Figure ( g ) shows a representative area digitally augmented from merged images of LC3 and cells stained with MitoTracker Red CMXRos. Places where both LC3 and mitochondria colocalize are observed in yellow.

    Techniques Used: Incubation, Immunofluorescence, Staining

    4) Product Images from "A testis-specific regulator of complex and hybrid N-glycan synthesis"

    Article Title: A testis-specific regulator of complex and hybrid N-glycan synthesis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201004102

    GnT1IP-L localizes to the ER, ERGIC, and Golgi. (A–C) HeLa cells transiently expressing different GnT1IP constructs were fixed, immunolabeled for Myc-tagged GnT1IP-L (red) and GM130 or PDI (green), or HA-tagged GnT1IP-L (green) and ERGIC-53 (red), followed by confocal microscopy (A and C) or fluorescence microscopy (B). (D) HeLa cells transiently expressing HA-GnT1IP-L or GlcNAcT-I-HA were treated with CHX for 45 min followed by BFA for 30 min, fixed, immunolabeled for HA-GnT1IP-L or GlcNAcT-I-HA (green) and ERGIC-53 (red), and analyzed by fluorescence microscopy. Bars, 10 µm.
    Figure Legend Snippet: GnT1IP-L localizes to the ER, ERGIC, and Golgi. (A–C) HeLa cells transiently expressing different GnT1IP constructs were fixed, immunolabeled for Myc-tagged GnT1IP-L (red) and GM130 or PDI (green), or HA-tagged GnT1IP-L (green) and ERGIC-53 (red), followed by confocal microscopy (A and C) or fluorescence microscopy (B). (D) HeLa cells transiently expressing HA-GnT1IP-L or GlcNAcT-I-HA were treated with CHX for 45 min followed by BFA for 30 min, fixed, immunolabeled for HA-GnT1IP-L or GlcNAcT-I-HA (green) and ERGIC-53 (red), and analyzed by fluorescence microscopy. Bars, 10 µm.

    Techniques Used: Expressing, Construct, Immunolabeling, Confocal Microscopy, Fluorescence, Microscopy

    5) Product Images from "Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles--a role of the endocytic-exocytic pathway"

    Article Title: Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles--a role of the endocytic-exocytic pathway

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201100147

    Association of prominin-1, but not CD34, with lipid raft-membrane vesicles released by HSPCs A. One-week-old co-culture HSPC/MSC conditioned medium was subjected to differential centrifugation for 5 min at 300 × g , 20 min at 1200 × g , 30 min at 10,000 × g , 60 min at 200,000 × g and 60 min at 400,000 × g . The resulting pellets were analysed by immunoblotting for either prominin-1 (top panel, arrow) or CD34 (bottom panel, arrowhead). Proteins in the 400,000 × g supernatant (Sup.) were analysed in parallel. B. The 200,000 × g pellet recovered after differential centrifugation (panel A) was subjected to equilibrium sucrose gradient (0.1–1.2 M) centrifugation. Equal volumes of the recovered fractions and the pellet were analysed by immunoblotting for prominin-1 (arrow). C,D. Negative EM analysis of the 200,000 × g pellet revealed the presence of small membrane vesicles (∼40–80 nm; arrows) and larger dense structures (∼200–600 nm; arrowhead). E. Negative staining EM of prominin-1 immunogold-labelled membrane vesicles recovered in the 200,000 × g pellet. Scale bars, 100 nm [(C–E)]. F. Haematopoietic cells (Cells) growing for 1 week in the co-culture system and 200,000 × g pellets (200,000 × g pellet) recovered upon differential centrifugation (panel A) were lysed for 30 min at 4°C in either 0.5% Triton X-100 or Lubrol WX or without detergent (PBS) and centrifuged for 60 min at 100,000 × g . The resulting supernatants (S) and pellets (P) were analysed by immunoblotting for prominin-1 (arrows). G. Lipid composition analysis of prominin-1-CMV was performed by C 14 -acetate labelling of HSPCs co-cultured with MSCs followed by TLC analysis of either haematopoietic cells (Cells) or the released prominin-1-positive vesicles recovered by a paramagnetic isolation with mAb CD133 (prominin-1-CMV). Percentages of cholesterol (Ch) and sphingomyelin (SM) of the total lipid composition are indicated. •, sample loading point.
    Figure Legend Snippet: Association of prominin-1, but not CD34, with lipid raft-membrane vesicles released by HSPCs A. One-week-old co-culture HSPC/MSC conditioned medium was subjected to differential centrifugation for 5 min at 300 × g , 20 min at 1200 × g , 30 min at 10,000 × g , 60 min at 200,000 × g and 60 min at 400,000 × g . The resulting pellets were analysed by immunoblotting for either prominin-1 (top panel, arrow) or CD34 (bottom panel, arrowhead). Proteins in the 400,000 × g supernatant (Sup.) were analysed in parallel. B. The 200,000 × g pellet recovered after differential centrifugation (panel A) was subjected to equilibrium sucrose gradient (0.1–1.2 M) centrifugation. Equal volumes of the recovered fractions and the pellet were analysed by immunoblotting for prominin-1 (arrow). C,D. Negative EM analysis of the 200,000 × g pellet revealed the presence of small membrane vesicles (∼40–80 nm; arrows) and larger dense structures (∼200–600 nm; arrowhead). E. Negative staining EM of prominin-1 immunogold-labelled membrane vesicles recovered in the 200,000 × g pellet. Scale bars, 100 nm [(C–E)]. F. Haematopoietic cells (Cells) growing for 1 week in the co-culture system and 200,000 × g pellets (200,000 × g pellet) recovered upon differential centrifugation (panel A) were lysed for 30 min at 4°C in either 0.5% Triton X-100 or Lubrol WX or without detergent (PBS) and centrifuged for 60 min at 100,000 × g . The resulting supernatants (S) and pellets (P) were analysed by immunoblotting for prominin-1 (arrows). G. Lipid composition analysis of prominin-1-CMV was performed by C 14 -acetate labelling of HSPCs co-cultured with MSCs followed by TLC analysis of either haematopoietic cells (Cells) or the released prominin-1-positive vesicles recovered by a paramagnetic isolation with mAb CD133 (prominin-1-CMV). Percentages of cholesterol (Ch) and sphingomyelin (SM) of the total lipid composition are indicated. •, sample loading point.

    Techniques Used: Co-Culture Assay, Centrifugation, Negative Staining, Cell Culture, Thin Layer Chromatography, Isolation

    Expression of prominin-1 versus either CD61 or CD42b A,B. Flow cytometric analysis revealed phenotypes of HSPCs cultured for 1 week on MSCs for the expression of prominin-1 (CD133) versus either CD61 (A) or CD42b (B). The gates were set according to unstained cells and isotype controls (left panels). The amount of CD133-bright, -dim and -negative cell population is shown as a percentage of the whole CD61- or CD42b-positive population (right panels). C. The percentage of cells harbouring the differentiation markers CD61 and CD42b increased concomitant with the decrease in prominin-1 (CD133) expression, resolving that they are mainly expressed on prominin-1-negative cell population. Data obtained from two independent experiments are shown.
    Figure Legend Snippet: Expression of prominin-1 versus either CD61 or CD42b A,B. Flow cytometric analysis revealed phenotypes of HSPCs cultured for 1 week on MSCs for the expression of prominin-1 (CD133) versus either CD61 (A) or CD42b (B). The gates were set according to unstained cells and isotype controls (left panels). The amount of CD133-bright, -dim and -negative cell population is shown as a percentage of the whole CD61- or CD42b-positive population (right panels). C. The percentage of cells harbouring the differentiation markers CD61 and CD42b increased concomitant with the decrease in prominin-1 (CD133) expression, resolving that they are mainly expressed on prominin-1-negative cell population. Data obtained from two independent experiments are shown.

    Techniques Used: Expressing, Flow Cytometry, Cell Culture

    6) Product Images from "The Putative Type III Secreted Chlamydia abortus Virulence-Associated Protein CAB063 Targets Lamin and Induces Apoptosis"

    Article Title: The Putative Type III Secreted Chlamydia abortus Virulence-Associated Protein CAB063 Targets Lamin and Induces Apoptosis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01059

    CAB063 co-localizes with lamin A along the host cell nuclear membrane. Co-localization (arrows) of CAB063 and lamin A is shown in pCI-CAB063 transfected and experimentally infected HeLa cells at 48 h post-infection and post-transfection, respectively. Goat-anti mouse AlexaFluor ®  647-conjugated secondary antibodies were used to visualize mouse-anti lamin A/C antibodies (pink), goat-anti rabbit AlexaFluor ®  488-conjugated secondary antibodies were used to visualize rabbit-anti CAB063 (green), DAPI was used for DNA staining (blue). Uninfected HeLa cells served as a control. Representative photographs out of  n  = 3 independent experiments are provided.
    Figure Legend Snippet: CAB063 co-localizes with lamin A along the host cell nuclear membrane. Co-localization (arrows) of CAB063 and lamin A is shown in pCI-CAB063 transfected and experimentally infected HeLa cells at 48 h post-infection and post-transfection, respectively. Goat-anti mouse AlexaFluor ® 647-conjugated secondary antibodies were used to visualize mouse-anti lamin A/C antibodies (pink), goat-anti rabbit AlexaFluor ® 488-conjugated secondary antibodies were used to visualize rabbit-anti CAB063 (green), DAPI was used for DNA staining (blue). Uninfected HeLa cells served as a control. Representative photographs out of n = 3 independent experiments are provided.

    Techniques Used: Transfection, Infection, Staining

    7) Product Images from "Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene"

    Article Title: Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01723-17

    Interactions between the Wood-Ljungdahl pathway, the Embden-Meyerhof-Parnas glycolytic pathway, and the mevalonic acid pathway for the synthesis of isoprene. For the Wood-Ljungdahl pathway: FDH, formate dehydrogenase, which also forms a complex with the electron bifurcating hydrogenase (EBH), HytA-E ( 21 ); Nfn, NADH-dependent ferredoxin(red)-NADP oxidoreductase ( 27 ); FTHFS, formyltetrahydrofolate synthetase; MTHFC, methenyltetrahydrofolate cyclohydrolase; MTHFD, NADP-dependent methylenetetrahydrofolate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase, putative electron-bifurcating complex ( 20 , 39 , but see 25 ); MET, methyltetrahydrofolate-corrinoid/iron-sulfur protein methyltransferase; CODH/ACS, bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase; PTA, phosphotransacetylase; ACK, acetate kinase. For the Embden-Meyerhof-Parnas (EMP) glycolytic pathway: FRUpts, fructose phosphoenolpyruvate-dependent phosphotransferase; HEX, hexokinase; PGI, phosphoglucose isomerase; PFK, phosphofructokinase, ATP- and PPi-dependent ( 36 ); FBA, fructose bisphosphate aldolase; DHAP, dihydroxyacetone phosphate; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde phosphate dehydrogenase, NAD and NADP-dependent ( 34 ); PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PEP, phosphoenolpyruvate; PYK, pyruvate kinase; PFOR, pyruvate ferredoxin oxidoreductase. For the mevalonic acid pathway: MvaE, acetyl-CoA acetyltransferase/HMG-CoA reductase, NADPH and NADH dependent ( 28 ); MvaS, hydroxymethylglutaryl-CoA synthase; Mvk, mevalonate kinase; Pmk, phosphomevalonate kinase; Mvd, mevalonate diphosphate decarboxylase; Idi, isopentenyl pyrophosphate isomerase; IspS, isoprene synthase; Rnf complex, 6-subunit membrane associated ion-motive complex ( 19 ). Reactions involving high-energy phosphate bonds are indicated with red arrows.
    Figure Legend Snippet: Interactions between the Wood-Ljungdahl pathway, the Embden-Meyerhof-Parnas glycolytic pathway, and the mevalonic acid pathway for the synthesis of isoprene. For the Wood-Ljungdahl pathway: FDH, formate dehydrogenase, which also forms a complex with the electron bifurcating hydrogenase (EBH), HytA-E ( 21 ); Nfn, NADH-dependent ferredoxin(red)-NADP oxidoreductase ( 27 ); FTHFS, formyltetrahydrofolate synthetase; MTHFC, methenyltetrahydrofolate cyclohydrolase; MTHFD, NADP-dependent methylenetetrahydrofolate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase, putative electron-bifurcating complex ( 20 , 39 , but see 25 ); MET, methyltetrahydrofolate-corrinoid/iron-sulfur protein methyltransferase; CODH/ACS, bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase; PTA, phosphotransacetylase; ACK, acetate kinase. For the Embden-Meyerhof-Parnas (EMP) glycolytic pathway: FRUpts, fructose phosphoenolpyruvate-dependent phosphotransferase; HEX, hexokinase; PGI, phosphoglucose isomerase; PFK, phosphofructokinase, ATP- and PPi-dependent ( 36 ); FBA, fructose bisphosphate aldolase; DHAP, dihydroxyacetone phosphate; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde phosphate dehydrogenase, NAD and NADP-dependent ( 34 ); PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PEP, phosphoenolpyruvate; PYK, pyruvate kinase; PFOR, pyruvate ferredoxin oxidoreductase. For the mevalonic acid pathway: MvaE, acetyl-CoA acetyltransferase/HMG-CoA reductase, NADPH and NADH dependent ( 28 ); MvaS, hydroxymethylglutaryl-CoA synthase; Mvk, mevalonate kinase; Pmk, phosphomevalonate kinase; Mvd, mevalonate diphosphate decarboxylase; Idi, isopentenyl pyrophosphate isomerase; IspS, isoprene synthase; Rnf complex, 6-subunit membrane associated ion-motive complex ( 19 ). Reactions involving high-energy phosphate bonds are indicated with red arrows.

    Techniques Used:

    8) Product Images from "Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes"

    Article Title: Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11289-8

    The immunogenicity of M2FA-lysine-BSA in the absence of adjuvant and anti-M2FA antibody titers in intact mice. C57BL/6 mice were injected i.p. with M2FA-lysine-BSA or BSA in the absence of adjuvant. The antibody titers of IgG ( a ) and IgM ( b ) against M2FA-lysine were detected using MFA-6ACA-KLH-coated plates. The anti-M2AA antibody titers were clearly increased in M2FA-lysine-BSA-immunized mice compared to the controls (BSA-treated mice). Values are mean and SD. ( c ) Intact female C57BL/6 mice (n = 4 or 5 per group) with different ages showed significantly different anti-M2FA IgG titers. Values are mean and SD. (*p
    Figure Legend Snippet: The immunogenicity of M2FA-lysine-BSA in the absence of adjuvant and anti-M2FA antibody titers in intact mice. C57BL/6 mice were injected i.p. with M2FA-lysine-BSA or BSA in the absence of adjuvant. The antibody titers of IgG ( a ) and IgM ( b ) against M2FA-lysine were detected using MFA-6ACA-KLH-coated plates. The anti-M2AA antibody titers were clearly increased in M2FA-lysine-BSA-immunized mice compared to the controls (BSA-treated mice). Values are mean and SD. ( c ) Intact female C57BL/6 mice (n = 4 or 5 per group) with different ages showed significantly different anti-M2FA IgG titers. Values are mean and SD. (*p

    Techniques Used: Mouse Assay, Injection

    Serum anti-M2FA IgG and IgM antibody levels in wild-type and ApoE −/− mice and immunohistochemical detection of M2FA-epitopes in heart valve of ApoE −/− mice. The anti-M2FA IgG ( a ) and IgM ( b ) antibody levels showed significant differences between wild-type and ApoE −/− mice with the M2FA-6ACA-BSA ELISAs (**p
    Figure Legend Snippet: Serum anti-M2FA IgG and IgM antibody levels in wild-type and ApoE −/− mice and immunohistochemical detection of M2FA-epitopes in heart valve of ApoE −/− mice. The anti-M2FA IgG ( a ) and IgM ( b ) antibody levels showed significant differences between wild-type and ApoE −/− mice with the M2FA-6ACA-BSA ELISAs (**p

    Techniques Used: Mouse Assay, Immunohistochemistry

    9) Product Images from "An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors"

    Article Title: An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s18010307

    ( a ) Schematic illustration showing the molecular structures formed by the surface functionalization on the IO nanostructure for binding the H1N1 subtype; ( b ) Reflectance peak positions for the pristine, APTMS-treated, NHS-PEG 4 -Maleimide cross linker-treated, and Cys-ProG -antibody immobilized IOs. Inset shows reflectance spectra for all four samples. APTMS: 3-aminopropyl trimethoxysilane.
    Figure Legend Snippet: ( a ) Schematic illustration showing the molecular structures formed by the surface functionalization on the IO nanostructure for binding the H1N1 subtype; ( b ) Reflectance peak positions for the pristine, APTMS-treated, NHS-PEG 4 -Maleimide cross linker-treated, and Cys-ProG -antibody immobilized IOs. Inset shows reflectance spectra for all four samples. APTMS: 3-aminopropyl trimethoxysilane.

    Techniques Used: Binding Assay

    10) Product Images from "Oxidative stress promotes exit from the stem cell state and spontaneous neuronal differentiation"

    Article Title: Oxidative stress promotes exit from the stem cell state and spontaneous neuronal differentiation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23786

    Enhanced ROS cause morphological changes in NT2 cells ( A ) Cells were treated with 25 μM paraquat for six days and immunostained with neuronal markers, NEUROD1 and TUJ1. The cells were counterstained with DAPI. The arrowhead denotes neurite-like cellular process. ( B, C ) Cells were treated with 100 μM paraquat ( B ) or 5 nM H 2 O 2 ( C ) for six days, and the morphological changes were visualized by phase-contrast microscopy. The arrowheads point to elongated cellular processes. Scale bar: 100 μm.
    Figure Legend Snippet: Enhanced ROS cause morphological changes in NT2 cells ( A ) Cells were treated with 25 μM paraquat for six days and immunostained with neuronal markers, NEUROD1 and TUJ1. The cells were counterstained with DAPI. The arrowhead denotes neurite-like cellular process. ( B, C ) Cells were treated with 100 μM paraquat ( B ) or 5 nM H 2 O 2 ( C ) for six days, and the morphological changes were visualized by phase-contrast microscopy. The arrowheads point to elongated cellular processes. Scale bar: 100 μm.

    Techniques Used: Microscopy

    MAPK-ERK1/2 signaling is activated by high ROS in NT2 cells ( A ) Cells were treated with the increasing concentrations of paraquat (PQ). The activation status of ERK1/2 and the upstream MEK1/2 was determined by immunoblotting with indicated antibodies. β-ACTIN was used as the loading control. ( B ) Cells were treated with indicated doses of MEK1/2 inhibitor, SL327. The efficacy of the inhibitor was demonstrated by immunoblotting for phospho-ERK1/2. ( C ) Cells were concurrently treated with 100 μM paraquat and indicated doses of SL327 for 6 days. The phase contrast images showed the cell density and morphology under different treatments. Scale bar: 100 µm.
    Figure Legend Snippet: MAPK-ERK1/2 signaling is activated by high ROS in NT2 cells ( A ) Cells were treated with the increasing concentrations of paraquat (PQ). The activation status of ERK1/2 and the upstream MEK1/2 was determined by immunoblotting with indicated antibodies. β-ACTIN was used as the loading control. ( B ) Cells were treated with indicated doses of MEK1/2 inhibitor, SL327. The efficacy of the inhibitor was demonstrated by immunoblotting for phospho-ERK1/2. ( C ) Cells were concurrently treated with 100 μM paraquat and indicated doses of SL327 for 6 days. The phase contrast images showed the cell density and morphology under different treatments. Scale bar: 100 µm.

    Techniques Used: Activation Assay

    Antioxidant signaling is activated by ROS and is involved in neuronal gene expression in NT2 cells ( A ) Cells were treated with the increasing concentrations of paraquat (PQ) for two days. The expression of the key redox signaling genes, Nrf2 and Keap1 , was measured by qPCR. ( B ) Cells were treated with paraquat alone (left panel) or co-treated with paraquat and GSH (right panel). The expression of the antioxidant Prdm16 mRNA level was examined by qPCR. ( C ) Cells were transfected with siRNA targeting Nrf2 (siNrf2) and then treated with paraquat for two days. The expression of neural progenitor marker ( Nestin ) and neuronal markers ( Grfa1 and Hoxa1 ) was monitored by qPCR. Bar: mean ± SD; * p
    Figure Legend Snippet: Antioxidant signaling is activated by ROS and is involved in neuronal gene expression in NT2 cells ( A ) Cells were treated with the increasing concentrations of paraquat (PQ) for two days. The expression of the key redox signaling genes, Nrf2 and Keap1 , was measured by qPCR. ( B ) Cells were treated with paraquat alone (left panel) or co-treated with paraquat and GSH (right panel). The expression of the antioxidant Prdm16 mRNA level was examined by qPCR. ( C ) Cells were transfected with siRNA targeting Nrf2 (siNrf2) and then treated with paraquat for two days. The expression of neural progenitor marker ( Nestin ) and neuronal markers ( Grfa1 and Hoxa1 ) was monitored by qPCR. Bar: mean ± SD; * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Marker

    Enhanced ROS decrease the expression of stemness genes in NT2 cells ( A ) Cells treated with paraquat (PQ) at the indicated concentrations were lyzed, and the protein levels of stemness markers, NANOG, OCT4 and TDGF1, were examined by Western blot. β-ACTIN was used as the loading control. Cells treated with indicated concentrations of PQ ( B ) or H 2 O 2 ( C ) for two days were lyzed for total RNAs. The transcript levels of stemness genes were measured by qPCR. Bar: mean ± SD; * p
    Figure Legend Snippet: Enhanced ROS decrease the expression of stemness genes in NT2 cells ( A ) Cells treated with paraquat (PQ) at the indicated concentrations were lyzed, and the protein levels of stemness markers, NANOG, OCT4 and TDGF1, were examined by Western blot. β-ACTIN was used as the loading control. Cells treated with indicated concentrations of PQ ( B ) or H 2 O 2 ( C ) for two days were lyzed for total RNAs. The transcript levels of stemness genes were measured by qPCR. Bar: mean ± SD; * p

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

    All-trans retinoic acid (atRA) induces neurogenic gene expression in NT2 cells Cells were treated with indicated concentrations of atRA for two days, and the transcript levels of canonical neuronal genes were determined by qPCR. Bar: mean ± SD; ** p
    Figure Legend Snippet: All-trans retinoic acid (atRA) induces neurogenic gene expression in NT2 cells Cells were treated with indicated concentrations of atRA for two days, and the transcript levels of canonical neuronal genes were determined by qPCR. Bar: mean ± SD; ** p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Paraquat induces the expression of neuronal markers in NT2 cells ( A ) Cells were treated with paraquat for different durations. qPCR was performed to measure the time-dependent expression of neuronal markers. ( B ) Cells were treated with indicated concentrations of paraquat for two days. The dose-dependent expression of neuronal transcription factors ( Pax6 and NeuroD1 ) and Cyp26a1 was quantified by qPCR. ( C ) Cells were treated with H 2 O 2 at different concentrations for two days and analysed by qPCR for the transcript levels of neuronal marker genes. Bar: mean ± SD; * p
    Figure Legend Snippet: Paraquat induces the expression of neuronal markers in NT2 cells ( A ) Cells were treated with paraquat for different durations. qPCR was performed to measure the time-dependent expression of neuronal markers. ( B ) Cells were treated with indicated concentrations of paraquat for two days. The dose-dependent expression of neuronal transcription factors ( Pax6 and NeuroD1 ) and Cyp26a1 was quantified by qPCR. ( C ) Cells were treated with H 2 O 2 at different concentrations for two days and analysed by qPCR for the transcript levels of neuronal marker genes. Bar: mean ± SD; * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Marker

    The antioxidant GSH suppresses the expression of neuronal markers induced by paraquat (PQ) ( A ) NT2 cells treated as indicated were collected for total RNA extraction. The expression of neuronal markers was measured by qPCR. ( B ) Cells were treated with atRA (RA) or co-treated with GSH for indicated days (D). The expression of neuronal markers was measured by qPCR. Bar: mean ± SD; * p
    Figure Legend Snippet: The antioxidant GSH suppresses the expression of neuronal markers induced by paraquat (PQ) ( A ) NT2 cells treated as indicated were collected for total RNA extraction. The expression of neuronal markers was measured by qPCR. ( B ) Cells were treated with atRA (RA) or co-treated with GSH for indicated days (D). The expression of neuronal markers was measured by qPCR. Bar: mean ± SD; * p

    Techniques Used: Expressing, RNA Extraction, Real-time Polymerase Chain Reaction

    Paraquat increases ROS level in NT2 cells ( A ) Cells were treated with paraquat at the indicated concentrations for 24 hours (left panel) and 40 hours (right panel). Chloromethyl-H2DCFDA dye was used to measure ROS level using spectrometry. Bar: mean ± SEM; ** p
    Figure Legend Snippet: Paraquat increases ROS level in NT2 cells ( A ) Cells were treated with paraquat at the indicated concentrations for 24 hours (left panel) and 40 hours (right panel). Chloromethyl-H2DCFDA dye was used to measure ROS level using spectrometry. Bar: mean ± SEM; ** p

    Techniques Used:

    11) Product Images from "Intracellular trafficking and cellular uptake mechanism of PHBV nanoparticles for targeted delivery in epithelial cell lines"

    Article Title: Intracellular trafficking and cellular uptake mechanism of PHBV nanoparticles for targeted delivery in epithelial cell lines

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-016-0241-6

    Determination of final nanoparticle fate in HeLa cells. HeLa cells were incubated with PHBV-RN nanoparticles for 15 min ( a – c ) and 1 h ( d – f ) at 37 °C; then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-LAMP1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm
    Figure Legend Snippet: Determination of final nanoparticle fate in HeLa cells. HeLa cells were incubated with PHBV-RN nanoparticles for 15 min ( a – c ) and 1 h ( d – f ) at 37 °C; then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-LAMP1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm

    Techniques Used: Incubation, Immunofluorescence, Fluorescence, Microscopy, Staining

    Evaluation of actin filaments rearrangement in the presence of PHBV nanoparticles. HeLa ( a – d ) and SKOV-3 ( e – h ) cells were incubated with PHBV-RN for 15 and 30 min at 37 °C; then cells were permeabilized and incubated with Phalloidin Alexa Fluor® 488 conjugated. Hoechst 33242 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm
    Figure Legend Snippet: Evaluation of actin filaments rearrangement in the presence of PHBV nanoparticles. HeLa ( a – d ) and SKOV-3 ( e – h ) cells were incubated with PHBV-RN for 15 and 30 min at 37 °C; then cells were permeabilized and incubated with Phalloidin Alexa Fluor® 488 conjugated. Hoechst 33242 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm

    Techniques Used: Incubation, Staining

    Characterization of the endocytosis mechanism using EEA-1 marker. HeLa ( a – c ) and SKOV-3 ( d – f ) cells were incubated with PHBV-RN nanoparticles for 15 min at 37 °C; then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-EEA1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm
    Figure Legend Snippet: Characterization of the endocytosis mechanism using EEA-1 marker. HeLa ( a – c ) and SKOV-3 ( d – f ) cells were incubated with PHBV-RN nanoparticles for 15 min at 37 °C; then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-EEA1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm

    Techniques Used: Marker, Incubation, Immunofluorescence, Fluorescence, Microscopy, Staining

    Determination of final nanoparticle fate in SKOV-3 cells. SKOV-3 cells were incubated with PHBV-RN nanoparticles for 15 min ( a – c ) and 1 h ( d – f ) at 37 °C, then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-LAMP1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm
    Figure Legend Snippet: Determination of final nanoparticle fate in SKOV-3 cells. SKOV-3 cells were incubated with PHBV-RN nanoparticles for 15 min ( a – c ) and 1 h ( d – f ) at 37 °C, then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-LAMP1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm

    Techniques Used: Incubation, Immunofluorescence, Fluorescence, Microscopy, Staining

    Characterization of the endocytosis mechanism using CAV-1 marker. HeLa ( a – c ) and SKOV-3 ( d – f ) cells were incubated with PHBV-RN nanoparticles for 15 min at 37 °C, then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-CAV1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm
    Figure Legend Snippet: Characterization of the endocytosis mechanism using CAV-1 marker. HeLa ( a – c ) and SKOV-3 ( d – f ) cells were incubated with PHBV-RN nanoparticles for 15 min at 37 °C, then cells were fixed and permeabilized. Later, an immunofluorescence was performed using anti-CAV1 (1:100) and anti-mouse Alexa Fluor® 488 (1:500) as primary and secondary antibody respectively. Finally, cells were observed through fluorescence microscopy. Hoechst 33342 was used as nuclear stain. Objective: ×60 magnification. Bar size 10 µm

    Techniques Used: Marker, Incubation, Immunofluorescence, Fluorescence, Microscopy, Staining

    12) Product Images from "Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets"

    Article Title: Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

    Journal: Beilstein Journal of Nanotechnology

    doi: 10.3762/bjnano.8.125

    Non-specific labelling of SC35 with Qdots. Fixed HeLa cells were dual labelled with green Alexa Fluor 488 (A) and red Qdot 625 (B) to produce an overlaid wide-field image (C). Scale bar is 20 μm. As a measure of co-localisation between Alexa Fluor 488 and Qdot 625, fluorescence intensities of the overlaid wide-field image (C) were analysed using a custom-written Matlab code to produce a correlation scatter plot (D). Pearson’s correlation coefficient ( r 2 ) of 0.006 indicates no co-localisation. The average Manders' coefficient of Alexa Fluor 488 overlapping with Qdot 625 (M1) was 0.19 (SD = 0.035, N = 3) and the average Manders’ coefficient of Qdot 625 overlapping with Alexa Fluor 488 (M2) was 0.08 (SD = 0.031, N = 3), as determined using JACoP.
    Figure Legend Snippet: Non-specific labelling of SC35 with Qdots. Fixed HeLa cells were dual labelled with green Alexa Fluor 488 (A) and red Qdot 625 (B) to produce an overlaid wide-field image (C). Scale bar is 20 μm. As a measure of co-localisation between Alexa Fluor 488 and Qdot 625, fluorescence intensities of the overlaid wide-field image (C) were analysed using a custom-written Matlab code to produce a correlation scatter plot (D). Pearson’s correlation coefficient ( r 2 ) of 0.006 indicates no co-localisation. The average Manders' coefficient of Alexa Fluor 488 overlapping with Qdot 625 (M1) was 0.19 (SD = 0.035, N = 3) and the average Manders’ coefficient of Qdot 625 overlapping with Alexa Fluor 488 (M2) was 0.08 (SD = 0.031, N = 3), as determined using JACoP.

    Techniques Used: Fluorescence

    Specific labelling of fibronectin with Qdots. Fixed rat mammary (Rama) 27 fibroblasts were dual labelled with green Alexa Fluor 488 (A) and a red Qdot 625 (B) to produce an overlaid wide-field image (C). Scale bar is 20 μm.
    Figure Legend Snippet: Specific labelling of fibronectin with Qdots. Fixed rat mammary (Rama) 27 fibroblasts were dual labelled with green Alexa Fluor 488 (A) and a red Qdot 625 (B) to produce an overlaid wide-field image (C). Scale bar is 20 μm.

    Techniques Used:

    Specific labelling of tubulin with Qdots. Methanol-fixed HeLa cells were labelled indirectly with a primary anti-tubulin antibody, green Alexa Fluor 488 (A), and red Qdot 625 (B), to produce an overlaid wide-field image (C). As a measure of co-localisation between Alexa Fluor 488 and Qdot 625, fluorescence intensities of the overlaid wide-field image (C) were analysed using a custom-written Matlab code to produce a correlation scatter plot (D). Pearson’s correlation coefficient ( r 2 ) of 0.93 indicates very high correlation. The average Manders coefficient of Alexa Fluor 488 overlapping with Qdot 625 (M1) was 0.99 (SD = 0.007, N = 3) and the average Manders coefficient of Qdot 625 overlapping with Alexa Fluor 488 (M2) was 0.99 (SD = 0.005, N = 3, as determined using JACoP. Paraformaldehyde fixed TC7 cells, expressing tubulin-GFP (E), were also labelled directly with an anti-GFP Qdot 625 conjugate (F) to produce an overlaid wide-field image (G,) and a corresponding correlation scatter plot (H); with a Pearson’s correlation coefficient ( r 2 ) of 0.86. Scale bar is 20 μm.
    Figure Legend Snippet: Specific labelling of tubulin with Qdots. Methanol-fixed HeLa cells were labelled indirectly with a primary anti-tubulin antibody, green Alexa Fluor 488 (A), and red Qdot 625 (B), to produce an overlaid wide-field image (C). As a measure of co-localisation between Alexa Fluor 488 and Qdot 625, fluorescence intensities of the overlaid wide-field image (C) were analysed using a custom-written Matlab code to produce a correlation scatter plot (D). Pearson’s correlation coefficient ( r 2 ) of 0.93 indicates very high correlation. The average Manders coefficient of Alexa Fluor 488 overlapping with Qdot 625 (M1) was 0.99 (SD = 0.007, N = 3) and the average Manders coefficient of Qdot 625 overlapping with Alexa Fluor 488 (M2) was 0.99 (SD = 0.005, N = 3, as determined using JACoP. Paraformaldehyde fixed TC7 cells, expressing tubulin-GFP (E), were also labelled directly with an anti-GFP Qdot 625 conjugate (F) to produce an overlaid wide-field image (G,) and a corresponding correlation scatter plot (H); with a Pearson’s correlation coefficient ( r 2 ) of 0.86. Scale bar is 20 μm.

    Techniques Used: Fluorescence, Expressing

    Non-specific labelling of talin with Qdots. Fixed HeLa cells were dual labelled with green Alexa Fluor 488 (A) and red Qdot 625 (B) to produce an overlaid wide-field image (C). Scale bar is 20 μm.
    Figure Legend Snippet: Non-specific labelling of talin with Qdots. Fixed HeLa cells were dual labelled with green Alexa Fluor 488 (A) and red Qdot 625 (B) to produce an overlaid wide-field image (C). Scale bar is 20 μm.

    Techniques Used:

    Strategies for immunofluorescence labelling. A fluorescent label (green) is conjugated to a secondary antibody (grey) or directly to a primary antibody (black), containing antigen-binding sites (red), which recognises and binds to a specific antigen (pink). Immunofluorescence labelling was either indirect with a primary antibody and a Qdot 625/Qdot 525 (Qdot-Ab) (A) or fluorescent dye such as Alexa Fluor 488/Cyanine 3 (Dye-Ab) (B) conjugated to a secondary antibody, or direct with an anti-GFP primary antibody conjugated to Qdot 625 (Qdot 625-GFP) (C). An alternative Qdot 625 was tried for indirect immunofluorescence labelling using a Qdot 625 streptavidin (yellow) conjugate (Qdot-Streptavidin) and biotinylated (blue) primary antibody (D). Scale bar is 10 nm.
    Figure Legend Snippet: Strategies for immunofluorescence labelling. A fluorescent label (green) is conjugated to a secondary antibody (grey) or directly to a primary antibody (black), containing antigen-binding sites (red), which recognises and binds to a specific antigen (pink). Immunofluorescence labelling was either indirect with a primary antibody and a Qdot 625/Qdot 525 (Qdot-Ab) (A) or fluorescent dye such as Alexa Fluor 488/Cyanine 3 (Dye-Ab) (B) conjugated to a secondary antibody, or direct with an anti-GFP primary antibody conjugated to Qdot 625 (Qdot 625-GFP) (C). An alternative Qdot 625 was tried for indirect immunofluorescence labelling using a Qdot 625 streptavidin (yellow) conjugate (Qdot-Streptavidin) and biotinylated (blue) primary antibody (D). Scale bar is 10 nm.

    Techniques Used: Immunofluorescence, Binding Assay

    13) Product Images from "A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma"

    Article Title: A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58507-4

    Characterization of the gene-edited oviductal cells. In ( a , b ) cell morphology of control (TP53 WT ) and edited cells (TP53 KO ) showing actin filaments (Phalloidin, green) nuclei (Hoechstt33342, blue) and tubulin (Acetylated tubulin, red); note the presence of ciliated cell (arrow) in TP53 WT ; and the presence of giant multi-nucleated cells (*) in TP53 KO . In ( c ) immunofluorescence for double-strand breaks (yH2AX, green) and cell proliferation (Ki67, green) and nuclei (Hoechst33342, blue); and the percentage of positive cells for those markers ( d ). * indicates that differences are statistically significant (paired sample t-test, p
    Figure Legend Snippet: Characterization of the gene-edited oviductal cells. In ( a , b ) cell morphology of control (TP53 WT ) and edited cells (TP53 KO ) showing actin filaments (Phalloidin, green) nuclei (Hoechstt33342, blue) and tubulin (Acetylated tubulin, red); note the presence of ciliated cell (arrow) in TP53 WT ; and the presence of giant multi-nucleated cells (*) in TP53 KO . In ( c ) immunofluorescence for double-strand breaks (yH2AX, green) and cell proliferation (Ki67, green) and nuclei (Hoechst33342, blue); and the percentage of positive cells for those markers ( d ). * indicates that differences are statistically significant (paired sample t-test, p

    Techniques Used: Immunofluorescence

    Fold change of relative mRNA expression of TP53 , Ki67 , BRCA1/2 , PAX8 , PTEN , My c and RB1 in TP53 KO vs . TP53 WT oviduct-on-a-chip platforms. * indicates statistically significant changes (paired samples t-test, p
    Figure Legend Snippet: Fold change of relative mRNA expression of TP53 , Ki67 , BRCA1/2 , PAX8 , PTEN , My c and RB1 in TP53 KO vs . TP53 WT oviduct-on-a-chip platforms. * indicates statistically significant changes (paired samples t-test, p

    Techniques Used: Expressing, Chromatin Immunoprecipitation

    Oviduct-on-a-chip model, leakage test and epithelial cell culture. In ( a ) different parts of the device with bottom (basolateral, pink) and top (apical, blue) compartments, and the assembled microfluidic device with a porous membrane in the middle; each device comprises two independent chambers. Right graphic shows a transverse cut of the chamber, indicating the oviductal epithelium growing on the top of the porous membrane in the apical compartment. In ( b ) leakage test showing the percentage of devices not leaking as a function of the applied shear stress; note lower shear stress used in brain- and liver-on-a-chip platforms 41 , 42 . In ( c ), immunofluorescence with reconstructed side-views of TP53 WT cells grown in the oviduct-on-a-chip for 2 weeks showing nuclei (Hoechst33342, blue), actin filaments (phalloidin, green), and cilia (acetylated alpha tubulin, red). In ( d ), relative mRNA expression of TP53, Ki67 and PAX8 of fresh collected cells and non-edited cells cultured in the oviduct-on-a-chip for 2 weeks. Scale bar = 25 µm.
    Figure Legend Snippet: Oviduct-on-a-chip model, leakage test and epithelial cell culture. In ( a ) different parts of the device with bottom (basolateral, pink) and top (apical, blue) compartments, and the assembled microfluidic device with a porous membrane in the middle; each device comprises two independent chambers. Right graphic shows a transverse cut of the chamber, indicating the oviductal epithelium growing on the top of the porous membrane in the apical compartment. In ( b ) leakage test showing the percentage of devices not leaking as a function of the applied shear stress; note lower shear stress used in brain- and liver-on-a-chip platforms 41 , 42 . In ( c ), immunofluorescence with reconstructed side-views of TP53 WT cells grown in the oviduct-on-a-chip for 2 weeks showing nuclei (Hoechst33342, blue), actin filaments (phalloidin, green), and cilia (acetylated alpha tubulin, red). In ( d ), relative mRNA expression of TP53, Ki67 and PAX8 of fresh collected cells and non-edited cells cultured in the oviduct-on-a-chip for 2 weeks. Scale bar = 25 µm.

    Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Immunofluorescence, Expressing

    Genetic manipulation of the oviductal cells. In ( a ), comparison of human and dog TP53 proteins, showing 79% homology in their sequence. ( b ), CRISPR-Cas9 mediated editing of TP53 gene in the oviduct-on-a-chip with a representation of all Insertions and Deletions (Indels) encountered by editing with both sgRNA 1 and 2 simultaneously. ( c ) depiction of the different Indels percentage. ( d ) section of Western blot for the TP53 gene in the WT and KO cells (full gel image and negative control presented as Supplementary Fig. S1 ). And ( e ) TP53 protein content normalized to B-actin (n = 4, paired sample t-tests, p = 0.024).
    Figure Legend Snippet: Genetic manipulation of the oviductal cells. In ( a ), comparison of human and dog TP53 proteins, showing 79% homology in their sequence. ( b ), CRISPR-Cas9 mediated editing of TP53 gene in the oviduct-on-a-chip with a representation of all Insertions and Deletions (Indels) encountered by editing with both sgRNA 1 and 2 simultaneously. ( c ) depiction of the different Indels percentage. ( d ) section of Western blot for the TP53 gene in the WT and KO cells (full gel image and negative control presented as Supplementary Fig. S1 ). And ( e ) TP53 protein content normalized to B-actin (n = 4, paired sample t-tests, p = 0.024).

    Techniques Used: Sequencing, CRISPR, Chromatin Immunoprecipitation, Western Blot, Negative Control

    14) Product Images from "Engineering a leucine zipper-TRAIL homotrimer with improved cytotoxicity in tumor cells"

    Article Title: Engineering a leucine zipper-TRAIL homotrimer with improved cytotoxicity in tumor cells

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-09-0202

    Cell surface expression of DR4/DR5 and DcR1/DcR2 in U251, PPC-1, and MCF7 tumor cells and human hepatocytes. A, Western blotting of DR4, DR5, DcR1, and DcR2. Cells were labeled with biotin and lysed. Labeled cell surface-associated DR4, DR5, DcR1, and
    Figure Legend Snippet: Cell surface expression of DR4/DR5 and DcR1/DcR2 in U251, PPC-1, and MCF7 tumor cells and human hepatocytes. A, Western blotting of DR4, DR5, DcR1, and DcR2. Cells were labeled with biotin and lysed. Labeled cell surface-associated DR4, DR5, DcR1, and

    Techniques Used: Expressing, Western Blot, Labeling

    15) Product Images from "Engineering a leucine zipper-TRAIL homotrimer with improved cytotoxicity in tumor cells"

    Article Title: Engineering a leucine zipper-TRAIL homotrimer with improved cytotoxicity in tumor cells

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-09-0202

    Cell surface expression of DR4/DR5 and DcR1/DcR2 in U251, PPC-1, and MCF7 tumor cells and human hepatocytes. A, Western blotting of DR4, DR5, DcR1, and DcR2. Cells were labeled with biotin and lysed. Labeled cell surface-associated DR4, DR5, DcR1, and
    Figure Legend Snippet: Cell surface expression of DR4/DR5 and DcR1/DcR2 in U251, PPC-1, and MCF7 tumor cells and human hepatocytes. A, Western blotting of DR4, DR5, DcR1, and DcR2. Cells were labeled with biotin and lysed. Labeled cell surface-associated DR4, DR5, DcR1, and

    Techniques Used: Expressing, Western Blot, Labeling

    16) Product Images from "Ehrlichia chaffeensis TRP120 Interacts with a Diverse Array of Eukaryotic Proteins Involved in Transcription, Signaling, and Cytoskeleton Organization ▿ TRP120 Interacts with a Diverse Array of Eukaryotic Proteins Involved in Transcription, Signaling, and Cytoskeleton Organization ▿ †"

    Article Title: Ehrlichia chaffeensis TRP120 Interacts with a Diverse Array of Eukaryotic Proteins Involved in Transcription, Signaling, and Cytoskeleton Organization ▿ TRP120 Interacts with a Diverse Array of Eukaryotic Proteins Involved in Transcription, Signaling, and Cytoskeleton Organization ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05608-11

    Colocalization of IGL, COX2, GGA1, ACTG1, and UNC13D with E. chaffeensis TRP120 in E. chaffeensis -infected THP-1 cells. Fluorescence microscopy and intensity profiles of infected THP-1 cells stained with 4,6′-diamidino-2-phenylindole (blue), anti-TRP120
    Figure Legend Snippet: Colocalization of IGL, COX2, GGA1, ACTG1, and UNC13D with E. chaffeensis TRP120 in E. chaffeensis -infected THP-1 cells. Fluorescence microscopy and intensity profiles of infected THP-1 cells stained with 4,6′-diamidino-2-phenylindole (blue), anti-TRP120

    Techniques Used: Infection, Fluorescence, Microscopy, Staining

    Colocalization of IGL, COX2, GGA1, PCGF5, ACTG1, and UNC13D with AcGFP-TRP120 in HeLa cells. pAcGFP1-TRP120-transfected HeLa cells (2 days posttransfection) were labeled and observed by fluorescence microscopy. The AcGFP-TRP120 (green; A to F) and anti-IGL,
    Figure Legend Snippet: Colocalization of IGL, COX2, GGA1, PCGF5, ACTG1, and UNC13D with AcGFP-TRP120 in HeLa cells. pAcGFP1-TRP120-transfected HeLa cells (2 days posttransfection) were labeled and observed by fluorescence microscopy. The AcGFP-TRP120 (green; A to F) and anti-IGL,

    Techniques Used: Transfection, Labeling, Fluorescence, Microscopy

    Confirmation of positive interactions in yeast by cotransformation. Y2HGold yeast cells were cotransformed with bait plasmid pGBKT7-TRP120 and prey plasmid pGADT7-IGL, -COX2, -GGA1, -PCGF5, -ACTG1, or -UNC13D. As a control, pGBKT7 (empty vector) was used
    Figure Legend Snippet: Confirmation of positive interactions in yeast by cotransformation. Y2HGold yeast cells were cotransformed with bait plasmid pGBKT7-TRP120 and prey plasmid pGADT7-IGL, -COX2, -GGA1, -PCGF5, -ACTG1, or -UNC13D. As a control, pGBKT7 (empty vector) was used

    Techniques Used: Plasmid Preparation

    E. chaffeensis TRP120 interactions with multiple human proteins detected by chemiluminescent coimmunoprecipitation. AcGFP1 (without insert) or AcGFP1-TRP120 was coexpressed with the PL-ACTG1, -GGA1, -PCGF5, or -UNC13D fusion protein in HeLa cells. A positive
    Figure Legend Snippet: E. chaffeensis TRP120 interactions with multiple human proteins detected by chemiluminescent coimmunoprecipitation. AcGFP1 (without insert) or AcGFP1-TRP120 was coexpressed with the PL-ACTG1, -GGA1, -PCGF5, or -UNC13D fusion protein in HeLa cells. A positive

    Techniques Used:

    17) Product Images from "Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells"

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/s12943-016-0547-x

    MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points
    Figure Legend Snippet: MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Techniques Used: Activity Assay, Stable Transfection, Expressing, Incubation, Imaging, Migration

    18) Product Images from "ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts"

    Article Title: ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200112107

    LPA-induced Rac activation in Y-27632–treated Swiss 3T3 cells. (A) Inhibition of membrane ruffles by expression of N17Rac in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pCMV5–N17Rac or 1 μg of pEGFP–C1. The transfected cells were cultured in DME containing 10% FBS for 16 h and then in serum-free DME for 24 h. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were stained for F-actin and phosphotyrosine as described in the legend to Fig. 1 . Arrows indicate transfected cells identified with anti-GFP (top) or anti-Flag staining (bottom). Note that membrane ruffles and focal complexes disappeared in the cells overexpressing N17Rac. Bar, 20 μm. (B) Pull-down assay for GTP-Rac. Swiss 3T3 cells were cultured and treated with either C3 exoenzyme or Y-27632 as described in the legend to Fig. 1 . The cells were then stimulated with 5 μM LPA for 0 and 5 min and subjected to the pull-down assay as described in Materials and methods. GTP-Rac precipitated from each cell lysates was analyzed by immunoblotting with anti-Rac antibody (top), and the total amounts of Rac present in the cell lysates are shown in the immunoblot in the bottom panels. −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that GTP-Rac increased in amount significantly by Y-27632 treatment and remained little in the C3 exoenzyme-treated cells.
    Figure Legend Snippet: LPA-induced Rac activation in Y-27632–treated Swiss 3T3 cells. (A) Inhibition of membrane ruffles by expression of N17Rac in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pCMV5–N17Rac or 1 μg of pEGFP–C1. The transfected cells were cultured in DME containing 10% FBS for 16 h and then in serum-free DME for 24 h. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were stained for F-actin and phosphotyrosine as described in the legend to Fig. 1 . Arrows indicate transfected cells identified with anti-GFP (top) or anti-Flag staining (bottom). Note that membrane ruffles and focal complexes disappeared in the cells overexpressing N17Rac. Bar, 20 μm. (B) Pull-down assay for GTP-Rac. Swiss 3T3 cells were cultured and treated with either C3 exoenzyme or Y-27632 as described in the legend to Fig. 1 . The cells were then stimulated with 5 μM LPA for 0 and 5 min and subjected to the pull-down assay as described in Materials and methods. GTP-Rac precipitated from each cell lysates was analyzed by immunoblotting with anti-Rac antibody (top), and the total amounts of Rac present in the cell lysates are shown in the immunoblot in the bottom panels. −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that GTP-Rac increased in amount significantly by Y-27632 treatment and remained little in the C3 exoenzyme-treated cells.

    Techniques Used: Activation Assay, Inhibition, Expressing, Transfection, Cell Culture, Staining, Pull Down Assay

    Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1 . The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.
    Figure Legend Snippet: Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1 . The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.

    Techniques Used: Inhibition, Cell Culture, Immunoprecipitation, Staining, Pull Down Assay, Expressing, Mutagenesis, Transfection

    Effects of C3 exoenzyme and Y-27632 on actin reorganization and localization of tyrosine-phosphorylated proteins in LPA-stimulated Swiss 3T3 cells. (A) Immunofluorescence. Swiss 3T3 cells were maintained in DME containing 10% FBS for 3 d and cultured in serum-free DME for 24 h. During this period, the cells were without any treatment or treated with either 30 μg/ml C3 exoenzyme for 4 d or with 30 μM Y-27632 for 30 min and then exposed to 5 μM LPA for 0 and 5 min. The cells were fixed, permeabilized, and stained with Texas red phalloidin for F-actin (red) and antiphosphotyrosine antibody (green). The top panels show the merged images of the cells without LPA stimulation. The F-actin staining, the phosphotyrosine staining, and the merged images of the LPA-stimulated cells are shown in the second, third, and the bottom rows of the panels, respectively. Note that the cells treated with Y-27632 display a thick rim of F-actin and dot-like phosphotyrosine staining in the cell periphery upon the addition of LPA. (B) Video microscopy. Swiss 3T3 cells transfected with GFP actin were serum starved for 24 h and treated with 30 μM Y-27632 for the last 30 min. LPA was added at 5 μM, and the cell shape change was monitored in the continued presence of Y-27632 by time-lapse confocal microscopy as the image of GFP actin. The number in each image indicates time after the LPA addition in min. See also the video available at http://www.jcb.org/cgi/content/full/jcb.200112107/DC1 . Bars, 20 μm.
    Figure Legend Snippet: Effects of C3 exoenzyme and Y-27632 on actin reorganization and localization of tyrosine-phosphorylated proteins in LPA-stimulated Swiss 3T3 cells. (A) Immunofluorescence. Swiss 3T3 cells were maintained in DME containing 10% FBS for 3 d and cultured in serum-free DME for 24 h. During this period, the cells were without any treatment or treated with either 30 μg/ml C3 exoenzyme for 4 d or with 30 μM Y-27632 for 30 min and then exposed to 5 μM LPA for 0 and 5 min. The cells were fixed, permeabilized, and stained with Texas red phalloidin for F-actin (red) and antiphosphotyrosine antibody (green). The top panels show the merged images of the cells without LPA stimulation. The F-actin staining, the phosphotyrosine staining, and the merged images of the LPA-stimulated cells are shown in the second, third, and the bottom rows of the panels, respectively. Note that the cells treated with Y-27632 display a thick rim of F-actin and dot-like phosphotyrosine staining in the cell periphery upon the addition of LPA. (B) Video microscopy. Swiss 3T3 cells transfected with GFP actin were serum starved for 24 h and treated with 30 μM Y-27632 for the last 30 min. LPA was added at 5 μM, and the cell shape change was monitored in the continued presence of Y-27632 by time-lapse confocal microscopy as the image of GFP actin. The number in each image indicates time after the LPA addition in min. See also the video available at http://www.jcb.org/cgi/content/full/jcb.200112107/DC1 . Bars, 20 μm.

    Techniques Used: Immunofluorescence, Cell Culture, Staining, Microscopy, Transfection, Confocal Microscopy

    19) Product Images from "ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts"

    Article Title: ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200112107

    LPA-induced Rac activation in Y-27632–treated Swiss 3T3 cells. (A) Inhibition of membrane ruffles by expression of N17Rac in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pCMV5–N17Rac or 1 μg of pEGFP–C1. The transfected cells were cultured in DME containing 10% FBS for 16 h and then in serum-free DME for 24 h. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were stained for F-actin and phosphotyrosine as described in the legend to Fig. 1 . Arrows indicate transfected cells identified with anti-GFP (top) or anti-Flag staining (bottom). Note that membrane ruffles and focal complexes disappeared in the cells overexpressing N17Rac. Bar, 20 μm. (B) Pull-down assay for GTP-Rac. Swiss 3T3 cells were cultured and treated with either C3 exoenzyme or Y-27632 as described in the legend to Fig. 1 . The cells were then stimulated with 5 μM LPA for 0 and 5 min and subjected to the pull-down assay as described in Materials and methods. GTP-Rac precipitated from each cell lysates was analyzed by immunoblotting with anti-Rac antibody (top), and the total amounts of Rac present in the cell lysates are shown in the immunoblot in the bottom panels. −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that GTP-Rac increased in amount significantly by Y-27632 treatment and remained little in the C3 exoenzyme-treated cells.
    Figure Legend Snippet: LPA-induced Rac activation in Y-27632–treated Swiss 3T3 cells. (A) Inhibition of membrane ruffles by expression of N17Rac in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pCMV5–N17Rac or 1 μg of pEGFP–C1. The transfected cells were cultured in DME containing 10% FBS for 16 h and then in serum-free DME for 24 h. The cells were treated with 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were stained for F-actin and phosphotyrosine as described in the legend to Fig. 1 . Arrows indicate transfected cells identified with anti-GFP (top) or anti-Flag staining (bottom). Note that membrane ruffles and focal complexes disappeared in the cells overexpressing N17Rac. Bar, 20 μm. (B) Pull-down assay for GTP-Rac. Swiss 3T3 cells were cultured and treated with either C3 exoenzyme or Y-27632 as described in the legend to Fig. 1 . The cells were then stimulated with 5 μM LPA for 0 and 5 min and subjected to the pull-down assay as described in Materials and methods. GTP-Rac precipitated from each cell lysates was analyzed by immunoblotting with anti-Rac antibody (top), and the total amounts of Rac present in the cell lysates are shown in the immunoblot in the bottom panels. −, cells without any pretreatment; Y, cells treated with Y-27632; C3, cells treated with C3 exoenzyme. Note that GTP-Rac increased in amount significantly by Y-27632 treatment and remained little in the C3 exoenzyme-treated cells.

    Techniques Used: Activation Assay, Inhibition, Expressing, Transfection, Cell Culture, Staining, Pull Down Assay

    Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1 . The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.
    Figure Legend Snippet: Inhibition of LPA-induced membrane ruffles by inhibiting tyrosine phosphorylation of Cas in Y-27632–treated cells. (A and B) Effects of PP1 on tyrosine phosphorylation of Cas and membrane ruffles induced by LPA. Swiss 3T3 cells were cultured and serum starved as described in the legend to Fig. 1 . The serum-starved cells were treated with either DMSO (control) or 50 μM PP1 (PP1) in the presence of 30 μM Y-27632 for 30 min and then stimulated with LPA for 5 min. The cells were then lysed for immunoprecipitation and immunoblotting analysis for tyrosine phosphorylation of Cas (A). Alternatively, the cells were fixed and stained for F-actin and phosphotyrosine as described above (B). Note that the PP1 treatment significantly inhibited Cas phosphorylation as shown by the immunoblot analysis and reduced the number of dot-like structures of tyrosine-phosphorylated proteins as illustrated by antiphosphotyrosine staining. Significant suppression of membrane ruffles is also noted in the PP1-treated cells. (C) Effects of PP1 on the level of GTP-Rac in Y-27632–treated cells. The cells were cultured and treated as described above and then subjected to the pull-down assay for GTP-Rac. Control, cells treated with Y-27632 alone; PP1, cells treated with both Y-27632 and PP1. (D) Inhibition of LPA-induced membrane ruffles by expression of CasΔSD, a tyrosine phosphorylation-defective Cas mutant, in Y-27632–treated cells. Swiss 3T3 cells were transfected with 1 μg of pSSRα–CasΔSD and cultured as described in the legend to Fig. 2 A. The cells were then treated with 30 μM Y-27632 for 30 min and stimulated with 5 μM LPA for 5 min in the continued presence of Y-27632. The cells were fixed, and F-actin and tyrosine-phosphorylated proteins were stained as described above. The cells expressing CasΔSD were identified by HA tag staining and are indicated by arrows. Bars, 20 μm.

    Techniques Used: Inhibition, Cell Culture, Immunoprecipitation, Staining, Pull Down Assay, Expressing, Mutagenesis, Transfection

    Effects of C3 exoenzyme and Y-27632 on actin reorganization and localization of tyrosine-phosphorylated proteins in LPA-stimulated Swiss 3T3 cells. (A) Immunofluorescence. Swiss 3T3 cells were maintained in DME containing 10% FBS for 3 d and cultured in serum-free DME for 24 h. During this period, the cells were without any treatment or treated with either 30 μg/ml C3 exoenzyme for 4 d or with 30 μM Y-27632 for 30 min and then exposed to 5 μM LPA for 0 and 5 min. The cells were fixed, permeabilized, and stained with Texas red phalloidin for F-actin (red) and antiphosphotyrosine antibody (green). The top panels show the merged images of the cells without LPA stimulation. The F-actin staining, the phosphotyrosine staining, and the merged images of the LPA-stimulated cells are shown in the second, third, and the bottom rows of the panels, respectively. Note that the cells treated with Y-27632 display a thick rim of F-actin and dot-like phosphotyrosine staining in the cell periphery upon the addition of LPA. (B) Video microscopy. Swiss 3T3 cells transfected with GFP actin were serum starved for 24 h and treated with 30 μM Y-27632 for the last 30 min. LPA was added at 5 μM, and the cell shape change was monitored in the continued presence of Y-27632 by time-lapse confocal microscopy as the image of GFP actin. The number in each image indicates time after the LPA addition in min. See also the video available at http://www.jcb.org/cgi/content/full/jcb.200112107/DC1 . Bars, 20 μm.
    Figure Legend Snippet: Effects of C3 exoenzyme and Y-27632 on actin reorganization and localization of tyrosine-phosphorylated proteins in LPA-stimulated Swiss 3T3 cells. (A) Immunofluorescence. Swiss 3T3 cells were maintained in DME containing 10% FBS for 3 d and cultured in serum-free DME for 24 h. During this period, the cells were without any treatment or treated with either 30 μg/ml C3 exoenzyme for 4 d or with 30 μM Y-27632 for 30 min and then exposed to 5 μM LPA for 0 and 5 min. The cells were fixed, permeabilized, and stained with Texas red phalloidin for F-actin (red) and antiphosphotyrosine antibody (green). The top panels show the merged images of the cells without LPA stimulation. The F-actin staining, the phosphotyrosine staining, and the merged images of the LPA-stimulated cells are shown in the second, third, and the bottom rows of the panels, respectively. Note that the cells treated with Y-27632 display a thick rim of F-actin and dot-like phosphotyrosine staining in the cell periphery upon the addition of LPA. (B) Video microscopy. Swiss 3T3 cells transfected with GFP actin were serum starved for 24 h and treated with 30 μM Y-27632 for the last 30 min. LPA was added at 5 μM, and the cell shape change was monitored in the continued presence of Y-27632 by time-lapse confocal microscopy as the image of GFP actin. The number in each image indicates time after the LPA addition in min. See also the video available at http://www.jcb.org/cgi/content/full/jcb.200112107/DC1 . Bars, 20 μm.

    Techniques Used: Immunofluorescence, Cell Culture, Staining, Microscopy, Transfection, Confocal Microscopy

    20) Product Images from "IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP"

    Article Title: IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP

    Journal: Nature Communications

    doi: 10.1038/ncomms14391

    Cytosolic DNA sensing and efficient innate signalling is dependent on IFI16. ( a ) Control and IFI16 CRISPR KO THP-1 cells were transfected with dsDNA at various concentrations and interferon induction measured after 6 h. ( b , c ) Control and IFI16 KO cells were transfected with dsDNA (4 μg ml −1 ) at indicated time-points ( b ) or poly (I:C) (1 μg ml −1 or 5 μg ml −1 ) for 20 h ( c ), hereafter supernatants were evaluated for type I interferon expression. ( d ) Whole cell lysates from control or IFI16 KO cells stimulated with dsDNA (4 μg ml −1 ) at indicated time-points were subjected to immunoblotting using antibodies against STING, pIRF3, pTBK1, total TBK, total IRF3 and vinculin (VCL) as loading control. ( e ) Control or IFI16 KO cells were transfected with dsDNA (4 μg ml −1 ) for two and four hours. The cells were fixed and stained with anti-IFI16 (Green) and anti-STING (Red) specific antibodies. DNA was visualized with DAPI (blue). Data represent mean±s.d. of biological triplicates, representative of three independent experiments. Unpaired t -test corrected for multiple comparisons using Holm–Sidak was been performed to evaluate the significance. * P
    Figure Legend Snippet: Cytosolic DNA sensing and efficient innate signalling is dependent on IFI16. ( a ) Control and IFI16 CRISPR KO THP-1 cells were transfected with dsDNA at various concentrations and interferon induction measured after 6 h. ( b , c ) Control and IFI16 KO cells were transfected with dsDNA (4 μg ml −1 ) at indicated time-points ( b ) or poly (I:C) (1 μg ml −1 or 5 μg ml −1 ) for 20 h ( c ), hereafter supernatants were evaluated for type I interferon expression. ( d ) Whole cell lysates from control or IFI16 KO cells stimulated with dsDNA (4 μg ml −1 ) at indicated time-points were subjected to immunoblotting using antibodies against STING, pIRF3, pTBK1, total TBK, total IRF3 and vinculin (VCL) as loading control. ( e ) Control or IFI16 KO cells were transfected with dsDNA (4 μg ml −1 ) for two and four hours. The cells were fixed and stained with anti-IFI16 (Green) and anti-STING (Red) specific antibodies. DNA was visualized with DAPI (blue). Data represent mean±s.d. of biological triplicates, representative of three independent experiments. Unpaired t -test corrected for multiple comparisons using Holm–Sidak was been performed to evaluate the significance. * P

    Techniques Used: CRISPR, Transfection, Expressing, Staining

    Recruitment of TBK1 to STING is dependent on IFI16 interactions. ( a ) Schematic illustration of the workflow of co-immunoprecipitation experiments. Cleared cell lysates (CCL) of THP-1 cells stimulated with dsDNA (4 μg ml −1 ) for 2 and 4 h were subjected to over-night co-immunoprecipitation with antibodies indicated in each panel. Lysates from control cells were co-IP with STING (lane 1–3) or IFI16 (lane 4–6). Input and elutes were analysed by gel electrophoresis followed by immunoblotting (IB) with the indicated antibodies. ( b ) STING co-IP samples from primary human MDMs after IB with the indicated antibodies. ( c ) STING co-IP samples from control (lane 1–3) and IFI16 KO (lane 4–6) THP-1 cells after IB with the indicated antibodies. ( d ) IFI16 co-IP samples from STING KO THP-1 cells after IB with the indicated antibodies. Each blot is representative of three independent experiments. ( e ) Control or IFI16 KO cells were stimulate with dsDNA (4 μg ml −1 ) for 2 h, fixed and stained for DAPI (blue), anti-IFI16 (green) or anti-IRF3 (red) and subjected to confocal imaging at × 63 oil lens. ( f ) Quantification of IRF3 localization of at least 50 individual cells treated as described in e .
    Figure Legend Snippet: Recruitment of TBK1 to STING is dependent on IFI16 interactions. ( a ) Schematic illustration of the workflow of co-immunoprecipitation experiments. Cleared cell lysates (CCL) of THP-1 cells stimulated with dsDNA (4 μg ml −1 ) for 2 and 4 h were subjected to over-night co-immunoprecipitation with antibodies indicated in each panel. Lysates from control cells were co-IP with STING (lane 1–3) or IFI16 (lane 4–6). Input and elutes were analysed by gel electrophoresis followed by immunoblotting (IB) with the indicated antibodies. ( b ) STING co-IP samples from primary human MDMs after IB with the indicated antibodies. ( c ) STING co-IP samples from control (lane 1–3) and IFI16 KO (lane 4–6) THP-1 cells after IB with the indicated antibodies. ( d ) IFI16 co-IP samples from STING KO THP-1 cells after IB with the indicated antibodies. Each blot is representative of three independent experiments. ( e ) Control or IFI16 KO cells were stimulate with dsDNA (4 μg ml −1 ) for 2 h, fixed and stained for DAPI (blue), anti-IFI16 (green) or anti-IRF3 (red) and subjected to confocal imaging at × 63 oil lens. ( f ) Quantification of IRF3 localization of at least 50 individual cells treated as described in e .

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Nucleic Acid Electrophoresis, Staining, Imaging

    IFI16 regulates cGAMP-mediated STING activation. ( a ) Control, IFI16, cGAS, STING KO THP-1 cells or ( b ) MDMs with IFI16 siRNA knockdown, were infused with cGAMP (50 nM) at indicated time-points and subsequently evaluated for type I interferon secretion. ( c ) STING dimerization analysis by semi-native western blotting. Upper lane represents an overexposure of the dimer STING band. Total STING was run on a separate SDS–Page gel. ( d ) Control and IFI16 KO cells were infused with cGAMP (50 nM) for 2 h, fixed and stained for DAPI (blue), IFI16 (green) and IRF3 (red). ( e ) IRF3 translocation from cytoplasm to nuclear saturation were quantified by counting > 50 separate images of control or IFI16 KO cells 2 h post cGAMP infusion. ( f ) Subcellular fractions of control and IFI16 KO cells stimulated with 50 nM cGAMP for 1 h were immunoblotted for phosphorylated IRF3 and total IRF3 in cytosolic (cyto) and nuclear (nucl) fractions. Data in ( a , b ) represent mean±s.d. of biological triplicates from ( a ) three independent experimental setups or ( b ) one donor; ( c – f ) data is representative of one of three independent experiments.
    Figure Legend Snippet: IFI16 regulates cGAMP-mediated STING activation. ( a ) Control, IFI16, cGAS, STING KO THP-1 cells or ( b ) MDMs with IFI16 siRNA knockdown, were infused with cGAMP (50 nM) at indicated time-points and subsequently evaluated for type I interferon secretion. ( c ) STING dimerization analysis by semi-native western blotting. Upper lane represents an overexposure of the dimer STING band. Total STING was run on a separate SDS–Page gel. ( d ) Control and IFI16 KO cells were infused with cGAMP (50 nM) for 2 h, fixed and stained for DAPI (blue), IFI16 (green) and IRF3 (red). ( e ) IRF3 translocation from cytoplasm to nuclear saturation were quantified by counting > 50 separate images of control or IFI16 KO cells 2 h post cGAMP infusion. ( f ) Subcellular fractions of control and IFI16 KO cells stimulated with 50 nM cGAMP for 1 h were immunoblotted for phosphorylated IRF3 and total IRF3 in cytosolic (cyto) and nuclear (nucl) fractions. Data in ( a , b ) represent mean±s.d. of biological triplicates from ( a ) three independent experimental setups or ( b ) one donor; ( c – f ) data is representative of one of three independent experiments.

    Techniques Used: Activation Assay, Western Blot, SDS Page, Staining, Translocation Assay

    21) Product Images from "Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine"

    Article Title: Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

    Journal: Scientific Reports

    doi: 10.1038/srep16479

    Promotion of enterocyte differentiation by combination treatment with three compounds and differentiation period extension. ( A ) The procedure for enterocyte differentiation from human iPS cells by treatment of compounds is presented. From day 19 to 24, the human iPS-derived intestinal cells were treated with the test compounds. ( B ) The gene expression leve ls of the enterocyte marker ANPEP in the test compound-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis on day 24. On the y axis, the gene expression levels in “Control (untreated hiPS-ELCs)” were taken as 1.0. ( C ) On day 24, the gene expression levels of the enterocyte marker VILLIN in the PMA, Wortmannin, SB431542, EGF or Wnt3A-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in “Control” were taken as 1.0. ( D ) Temporal gene expression levels of ANPEP in the human iPS cell-derived intestinal cells (day 24, 29, and 34) were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Adult Intestine were taken as 1.0. ( E ) The modified enterocyte differentiation protocol is illustrated. ( F ) A morphological image of human iPS-derived enterocyte-like cells is represented. Scale bar represents 100 μm. ( G ) Human iPS cell-derived enterocyte-like cells were assayed for the expression of intestinal marker CDX2 (Red) by immunohistochemistry. Nuclei were stained with DAPI (Blue). Scale bar represents 40 μm. ( H ) Percentages of VILLIN-positive cells in the SB431542, EGF, and Wnt3A-treated enterocyte-like cells were analyzed by flow cytometry analysis on day 24 and 34. Data are represented as the means ± S.E. ( n ≧ 3). Statistical analysis was performed using the unpaired two-tailed student’s t -test. * P
    Figure Legend Snippet: Promotion of enterocyte differentiation by combination treatment with three compounds and differentiation period extension. ( A ) The procedure for enterocyte differentiation from human iPS cells by treatment of compounds is presented. From day 19 to 24, the human iPS-derived intestinal cells were treated with the test compounds. ( B ) The gene expression leve ls of the enterocyte marker ANPEP in the test compound-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis on day 24. On the y axis, the gene expression levels in “Control (untreated hiPS-ELCs)” were taken as 1.0. ( C ) On day 24, the gene expression levels of the enterocyte marker VILLIN in the PMA, Wortmannin, SB431542, EGF or Wnt3A-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in “Control” were taken as 1.0. ( D ) Temporal gene expression levels of ANPEP in the human iPS cell-derived intestinal cells (day 24, 29, and 34) were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Adult Intestine were taken as 1.0. ( E ) The modified enterocyte differentiation protocol is illustrated. ( F ) A morphological image of human iPS-derived enterocyte-like cells is represented. Scale bar represents 100 μm. ( G ) Human iPS cell-derived enterocyte-like cells were assayed for the expression of intestinal marker CDX2 (Red) by immunohistochemistry. Nuclei were stained with DAPI (Blue). Scale bar represents 40 μm. ( H ) Percentages of VILLIN-positive cells in the SB431542, EGF, and Wnt3A-treated enterocyte-like cells were analyzed by flow cytometry analysis on day 24 and 34. Data are represented as the means ± S.E. ( n ≧ 3). Statistical analysis was performed using the unpaired two-tailed student’s t -test. * P

    Techniques Used: Derivative Assay, Expressing, Marker, Quantitative RT-PCR, Modification, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Two Tailed Test

    22) Product Images from "Heparan Sulfate Expression in the Neural Crest is Essential for Mouse Cardiogenesis"

    Article Title: Heparan Sulfate Expression in the Neural Crest is Essential for Mouse Cardiogenesis

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2013.10.013

    2.1 Heart defects in NDST1 deficient embryos
    Figure Legend Snippet: 2.1 Heart defects in NDST1 deficient embryos

    Techniques Used:

    2.2 NDST1 expression and HS composition in the mouse embryonic heart
    Figure Legend Snippet: 2.2 NDST1 expression and HS composition in the mouse embryonic heart

    Techniques Used: Expressing

    NDST1 mutation disrupted FGF/FGFR interaction
    Figure Legend Snippet: NDST1 mutation disrupted FGF/FGFR interaction

    Techniques Used: Mutagenesis

    Heart defects in NDST1 mutant E18.5 embryos
    Figure Legend Snippet: Heart defects in NDST1 mutant E18.5 embryos

    Techniques Used: Mutagenesis

    Altered HS structure and loss of Ptc expression in NDST1 mutant valves
    Figure Legend Snippet: Altered HS structure and loss of Ptc expression in NDST1 mutant valves

    Techniques Used: Expressing, Mutagenesis

    Neural crest cell specific knockout of NDST1 results in VSD and OFT defects
    Figure Legend Snippet: Neural crest cell specific knockout of NDST1 results in VSD and OFT defects

    Techniques Used: Knock-Out

    2.2 NDST1 expression and HS composition in the mouse embryonic heart
    Figure Legend Snippet: 2.2 NDST1 expression and HS composition in the mouse embryonic heart

    Techniques Used: Expressing

    Loss of FGF signaling and impaired proliferation in NDST1 −/− ventricular septa
    Figure Legend Snippet: Loss of FGF signaling and impaired proliferation in NDST1 −/− ventricular septa

    Techniques Used:

    23) Product Images from "Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2 ▿Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2 ▿ †"

    Article Title: Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2 ▿Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2 ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01164-09

    Mapping the STAT2 interaction domain to the carboxy-terminal region of the hCMV IE1 protein. (A) Schematic overview of the tested wild-type and mutant CMV major IE proteins (drawn to scale). Black bars correspond to identical sequences in the respective IE1 and IE2 proteins. Black lines symbolize unique sequences in the IE2 and mIE1 proteins. The STAT2 region interacting within IE1, deduced from the experiments whose results are shown in panels C and D, is shown as a white bar at the top of the diagram. NLS, IE1 nuclear localization signal (locations of IE2- and mIE1-specific nuclear localization signals are not shown); CTD, chromatin-tethering domain. (B) Purified proteins used for GST pull-down assays. Following affinity purification, the indicated GST and GST-IE proteins were separated in 10% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. (C) Results of pull-down binding assays. Identical volumes of glutathione Sepharose beads carrying equal amounts of GST or the indicated GST-IE fusion proteins were reacted with lysis buffer or whole-cell lysate from MRC-5 fibroblasts. Samples were separated in 10% polyacrylamide-SDS gels and Western blotted. Input and copurified STAT2 was detected using antibody H-190. In the GST-mIE1 lane (lower right panel), STAT2 (113 kDa) was detected at an apparently lower molecular weight compared to the other lanes. This is likely caused by comigrating excess GST-mIE1 (∼115 kDa) affecting STAT2 mobility. (D) Nuclear distribution of ectopically expressed EGFP-IE proteins and endogenous STAT2 in 2fTGH cells. After plasmid transfection and short selection in G418 (300 μg/ml), cells were treated with 1,000 U IFN-α for 1 h and fixed with methanol. Then samples were simultaneously reacted with primary antibodies against STAT2 (H-190) and EGFP (MAB3580), followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate, a mouse-specific Alexa Fluor 488 conjugate, and DAPI. Representative single and merged fluorescent stainings of three different subcellular localization patterns are shown: mitotic (M), interphase with nuclear punctate (and diffuse) IE protein distribution (IP), and interphase with nuclear diffuse protein staining (ID). Since EGFP and EGFP-IE1ΔN do not localize to nuclear dots, only M and ID patterns are shown in the upper two sets of images. Magnification, ∼×500.
    Figure Legend Snippet: Mapping the STAT2 interaction domain to the carboxy-terminal region of the hCMV IE1 protein. (A) Schematic overview of the tested wild-type and mutant CMV major IE proteins (drawn to scale). Black bars correspond to identical sequences in the respective IE1 and IE2 proteins. Black lines symbolize unique sequences in the IE2 and mIE1 proteins. The STAT2 region interacting within IE1, deduced from the experiments whose results are shown in panels C and D, is shown as a white bar at the top of the diagram. NLS, IE1 nuclear localization signal (locations of IE2- and mIE1-specific nuclear localization signals are not shown); CTD, chromatin-tethering domain. (B) Purified proteins used for GST pull-down assays. Following affinity purification, the indicated GST and GST-IE proteins were separated in 10% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. (C) Results of pull-down binding assays. Identical volumes of glutathione Sepharose beads carrying equal amounts of GST or the indicated GST-IE fusion proteins were reacted with lysis buffer or whole-cell lysate from MRC-5 fibroblasts. Samples were separated in 10% polyacrylamide-SDS gels and Western blotted. Input and copurified STAT2 was detected using antibody H-190. In the GST-mIE1 lane (lower right panel), STAT2 (113 kDa) was detected at an apparently lower molecular weight compared to the other lanes. This is likely caused by comigrating excess GST-mIE1 (∼115 kDa) affecting STAT2 mobility. (D) Nuclear distribution of ectopically expressed EGFP-IE proteins and endogenous STAT2 in 2fTGH cells. After plasmid transfection and short selection in G418 (300 μg/ml), cells were treated with 1,000 U IFN-α for 1 h and fixed with methanol. Then samples were simultaneously reacted with primary antibodies against STAT2 (H-190) and EGFP (MAB3580), followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate, a mouse-specific Alexa Fluor 488 conjugate, and DAPI. Representative single and merged fluorescent stainings of three different subcellular localization patterns are shown: mitotic (M), interphase with nuclear punctate (and diffuse) IE protein distribution (IP), and interphase with nuclear diffuse protein staining (ID). Since EGFP and EGFP-IE1ΔN do not localize to nuclear dots, only M and ID patterns are shown in the upper two sets of images. Magnification, ∼×500.

    Techniques Used: Mutagenesis, Purification, Affinity Purification, Staining, Binding Assay, Lysis, Western Blot, Molecular Weight, Plasmid Preparation, Transfection, Selection, Incubation

    Relative contributions of IE1 LC motifs to subnuclear colocalization with STAT2. (A) Nuclear distribution of ectopically expressed wild-type and mutant EGFP-IE1 proteins and endogenous STAT2 in 2fTGH cells. After plasmid transfection and short selection in G418 (300 μg/ml), cells were treated with 1,000 U IFN-α for 1 h and fixed with methanol. Then samples were simultaneously reacted with primary antibodies against STAT2 (H-190) and EGFP (MAB3580), followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate, a mouse-specific Alexa Fluor 488 conjugate, and DAPI. Representative single and merged fluorescent stainings of three different subcellular localization patterns are shown: mitotic (M), interphase with predominantly nuclear punctate IE1 distribution (IP), and interphase with nuclear diffuse protein staining (ID). Magnification, ∼×500. (B) Quantitation of IE1-dependent STAT2 sequestration at ND10. At least 100 nuclei with punctate staining of wild-type or mutant IE1 were analyzed for codistribution of STAT2 by immunofluorescence microscopy, as shown in panel A. Results are percentages of full-length-IE1 activity (set to 100%). For comparison, semiquantitative results from immunofluorescent colocalizations between IE1 and STAT2 at mitotic chromatin are also shown: +++, wild-type activity; ++, moderately reduced activity; +, severely reduced activity; −, no detectable activity.
    Figure Legend Snippet: Relative contributions of IE1 LC motifs to subnuclear colocalization with STAT2. (A) Nuclear distribution of ectopically expressed wild-type and mutant EGFP-IE1 proteins and endogenous STAT2 in 2fTGH cells. After plasmid transfection and short selection in G418 (300 μg/ml), cells were treated with 1,000 U IFN-α for 1 h and fixed with methanol. Then samples were simultaneously reacted with primary antibodies against STAT2 (H-190) and EGFP (MAB3580), followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate, a mouse-specific Alexa Fluor 488 conjugate, and DAPI. Representative single and merged fluorescent stainings of three different subcellular localization patterns are shown: mitotic (M), interphase with predominantly nuclear punctate IE1 distribution (IP), and interphase with nuclear diffuse protein staining (ID). Magnification, ∼×500. (B) Quantitation of IE1-dependent STAT2 sequestration at ND10. At least 100 nuclei with punctate staining of wild-type or mutant IE1 were analyzed for codistribution of STAT2 by immunofluorescence microscopy, as shown in panel A. Results are percentages of full-length-IE1 activity (set to 100%). For comparison, semiquantitative results from immunofluorescent colocalizations between IE1 and STAT2 at mitotic chromatin are also shown: +++, wild-type activity; ++, moderately reduced activity; +, severely reduced activity; −, no detectable activity.

    Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Selection, Incubation, Staining, Quantitation Assay, Immunofluorescence, Microscopy, Activity Assay

    24) Product Images from "Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB"

    Article Title: Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12855

    Quantitative gene expression analysis of CT009, cell replication, and cell division encoding components
    Figure Legend Snippet: Quantitative gene expression analysis of CT009, cell replication, and cell division encoding components

    Techniques Used: Expressing

    Qualitative β-galactosidase activity reflecting CT009 protein interactions with FtsK and RodA
    Figure Legend Snippet: Qualitative β-galactosidase activity reflecting CT009 protein interactions with FtsK and RodA

    Techniques Used: Activity Assay

    1.25 Å crystal Structure of CT009 from C. trachomatis adopts an “open” fold, resulting in a subdomain swap dimer
    Figure Legend Snippet: 1.25 Å crystal Structure of CT009 from C. trachomatis adopts an “open” fold, resulting in a subdomain swap dimer

    Techniques Used:

    Crystallographic dimer of CT009 has structural similarity to RodZ from T. maritima , including conservation of key MreB-interacting residues
    Figure Legend Snippet: Crystallographic dimer of CT009 has structural similarity to RodZ from T. maritima , including conservation of key MreB-interacting residues

    Techniques Used:

    CT009 is able to partially complement morphology of rodZ deficient E. coli
    Figure Legend Snippet: CT009 is able to partially complement morphology of rodZ deficient E. coli

    Techniques Used:

    Localization of CT009 or MreB within C. trachomatis
    Figure Legend Snippet: Localization of CT009 or MreB within C. trachomatis

    Techniques Used:

    Predicted domain organization of RodZ and CT009 and computational models of predicted protein structure
    Figure Legend Snippet: Predicted domain organization of RodZ and CT009 and computational models of predicted protein structure

    Techniques Used:

    25) Product Images from "Epstein–Barr Virus Lytic Reactivation Induces IgG4 Production by Host B Lymphocytes in Graves' Disease Patients and Controls: A Subset of Graves' Disease Is an IgG4-Related Disease-Like Condition"

    Article Title: Epstein–Barr Virus Lytic Reactivation Induces IgG4 Production by Host B Lymphocytes in Graves' Disease Patients and Controls: A Subset of Graves' Disease Is an IgG4-Related Disease-Like Condition

    Journal: Viral Immunology

    doi: 10.1089/vim.2018.0042

    EBER1-positive cells and IgG4-positive cells were observed in the same areas. Resected tissues showed the diffuse hyperplasia of thyroid follicular epithelial cells with the focal infiltration of lymphocytes, but not tumefactive lesions, storiform fibrosis, or obliterative phlebitis (A) . EBER1-positive cells and IgG4-positive plasma cells were observed in the same area with lymphoid cell infiltration (B, D) . Six of the seven cases had a large number of IgG4-positive plasma cells (10/HPF
    Figure Legend Snippet: EBER1-positive cells and IgG4-positive cells were observed in the same areas. Resected tissues showed the diffuse hyperplasia of thyroid follicular epithelial cells with the focal infiltration of lymphocytes, but not tumefactive lesions, storiform fibrosis, or obliterative phlebitis (A) . EBER1-positive cells and IgG4-positive plasma cells were observed in the same area with lymphoid cell infiltration (B, D) . Six of the seven cases had a large number of IgG4-positive plasma cells (10/HPF

    Techniques Used:

    Production of IgG and IgG4 during the EBV reactivation period. Culture fluids were sampled on days 0, 5, 10, and 12, and IgG (A) and IgG4 (B) were then measured by ELISA. Time course changes were significant in Friedman's analysis of variance. IgG4 percentages (C) were higher than normal serum levels (approximately 4%). However, no significant difference was observed between patients and controls (D) .
    Figure Legend Snippet: Production of IgG and IgG4 during the EBV reactivation period. Culture fluids were sampled on days 0, 5, 10, and 12, and IgG (A) and IgG4 (B) were then measured by ELISA. Time course changes were significant in Friedman's analysis of variance. IgG4 percentages (C) were higher than normal serum levels (approximately 4%). However, no significant difference was observed between patients and controls (D) .

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of IgG4(+)72A1(+) double-positive cells in culture cells on day 5. We detected IgG4-positive and EBV-reactivated [IgG4(+)72A1(+)] cells in culture cells on day 5 and confirmed sorted cells by confocal laser microscope. Red spots are surface IgG4 and fine green dots ).
    Figure Legend Snippet: Detection of IgG4(+)72A1(+) double-positive cells in culture cells on day 5. We detected IgG4-positive and EBV-reactivated [IgG4(+)72A1(+)] cells in culture cells on day 5 and confirmed sorted cells by confocal laser microscope. Red spots are surface IgG4 and fine green dots ).

    Techniques Used: Microscopy

    26) Product Images from "Substrate Cleavage and Duration of Action of Botulinum Neurotoxin Type FA (“H, HA”)"

    Article Title: Substrate Cleavage and Duration of Action of Botulinum Neurotoxin Type FA (“H, HA”)

    Journal: Toxicon : official journal of the International Society on Toxinology

    doi: 10.1016/j.toxicon.2017.12.048

    Estimation of the half-life of BoNT/FA, /B1, /F1, and /F5 LCs in hiPSC derived neurons. The hiPSC derived neurons were exposed to serial dilutions of each toxin for 48 h, and all extracellular toxin was removed. Cells continued to be incubated at 37°C in fresh culture media. At the indicated time points, triplicate sets of cells exposed to the dilution series were harvested and analysed for VAMP2 cleavage by Western blot. The top panel shows graphs depicting the average and standard deviations of triplicate samples at each time point, and nonlinear regression fits to determine the EC50 values (Prism 6 software), respectively. The bottom panel shows XY scatter blots of the EC50 values versus time and an exponential trendline fitted through the EC50 values. The half-life for recovery was determined from the slope of the trendline using the formula t1/2=LN(2)/slope. Note: The EC50 value at day 7 for BoNT/F5 was estimated by curve projection, since the low activity of BoNT/F5 combined with low recoveries from purifications limited the doses that could be used in this assay.
    Figure Legend Snippet: Estimation of the half-life of BoNT/FA, /B1, /F1, and /F5 LCs in hiPSC derived neurons. The hiPSC derived neurons were exposed to serial dilutions of each toxin for 48 h, and all extracellular toxin was removed. Cells continued to be incubated at 37°C in fresh culture media. At the indicated time points, triplicate sets of cells exposed to the dilution series were harvested and analysed for VAMP2 cleavage by Western blot. The top panel shows graphs depicting the average and standard deviations of triplicate samples at each time point, and nonlinear regression fits to determine the EC50 values (Prism 6 software), respectively. The bottom panel shows XY scatter blots of the EC50 values versus time and an exponential trendline fitted through the EC50 values. The half-life for recovery was determined from the slope of the trendline using the formula t1/2=LN(2)/slope. Note: The EC50 value at day 7 for BoNT/F5 was estimated by curve projection, since the low activity of BoNT/F5 combined with low recoveries from purifications limited the doses that could be used in this assay.

    Techniques Used: Derivative Assay, Incubation, Western Blot, Software, Activity Assay

    Potency and VAMP cleavage of BoNT/FA in primary mouse spinal cord (MSC) cells. A: Western blot data of BoNT/FA potency in MSC cells. MSC cells were exposed to serial dilutions of BoNT/FA for 48 h, and cell lysates were analysed for VAMP1 and VAMP2 cleavage by Western blot and densitometry. Each toxin dilution was tested in triplicate, and average and standard deviation are shown. Prism 6 was used to generate a non-linear regression fit and estimate the EC50 values for VAMP1 and 2 cleavage, respectively. 95 % confidence intervals for EC50s were 0.03–0.2 (VAMP2) and 0.005–0.01 (VAMP1) B: Immunocytochemistry of MSC cells to determine VAMP1 (left panel) and VAMP2 (right panel) expression patterns. VAMP1 or 2 are shown in green, MAP2 staining of neurons is shown in red, and GFAP staining of glial cells is shown in blue. C: In vitro analysis of VAMP1 and 2 cleavage in MSC cell lysates. MSC cells lysates were prepared and incubated with the indicated concentrations of BoNT/FA, /F1, or /B1 for 30 min at 37°C after reduction of the toxin s with DTT. VAMP1 and 2 cleavage was determined by Western blot and densitometry, and the average and standard deviation of triplicate samples is shown, respectively. Prism 6 was used to generate a non-linear regression fit and estimate the EC50 values for VAMP1 and 2 cleavage, respectively. 95 % confidence intervals for the EC50 estimates were: BoNT/FA: 0.15–0.21 (VAMP1), 0.16–0.3 (VAMP2), BoNT/F1: 1.2–1.9 (VAMP1), 2.07–2.1 (VAMP2), BoNT/B1: 1.2–2.7 (VAMP1), 9.5–28.2 (VAMP2).
    Figure Legend Snippet: Potency and VAMP cleavage of BoNT/FA in primary mouse spinal cord (MSC) cells. A: Western blot data of BoNT/FA potency in MSC cells. MSC cells were exposed to serial dilutions of BoNT/FA for 48 h, and cell lysates were analysed for VAMP1 and VAMP2 cleavage by Western blot and densitometry. Each toxin dilution was tested in triplicate, and average and standard deviation are shown. Prism 6 was used to generate a non-linear regression fit and estimate the EC50 values for VAMP1 and 2 cleavage, respectively. 95 % confidence intervals for EC50s were 0.03–0.2 (VAMP2) and 0.005–0.01 (VAMP1) B: Immunocytochemistry of MSC cells to determine VAMP1 (left panel) and VAMP2 (right panel) expression patterns. VAMP1 or 2 are shown in green, MAP2 staining of neurons is shown in red, and GFAP staining of glial cells is shown in blue. C: In vitro analysis of VAMP1 and 2 cleavage in MSC cell lysates. MSC cells lysates were prepared and incubated with the indicated concentrations of BoNT/FA, /F1, or /B1 for 30 min at 37°C after reduction of the toxin s with DTT. VAMP1 and 2 cleavage was determined by Western blot and densitometry, and the average and standard deviation of triplicate samples is shown, respectively. Prism 6 was used to generate a non-linear regression fit and estimate the EC50 values for VAMP1 and 2 cleavage, respectively. 95 % confidence intervals for the EC50 estimates were: BoNT/FA: 0.15–0.21 (VAMP1), 0.16–0.3 (VAMP2), BoNT/F1: 1.2–1.9 (VAMP1), 2.07–2.1 (VAMP2), BoNT/B1: 1.2–2.7 (VAMP1), 9.5–28.2 (VAMP2).

    Techniques Used: Western Blot, Standard Deviation, Immunocytochemistry, Expressing, Staining, In Vitro, Incubation

    27) Product Images from "Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183"

    Article Title: Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190407

    Characterization of the novel ubiquitin ligase RNF183. ]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin ( upper panel) and anti-V5 antibodies ( lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 ( red ) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green ) and DAPI for nuclear staining ( blue ).
    Figure Legend Snippet: Characterization of the novel ubiquitin ligase RNF183. ]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin ( upper panel) and anti-V5 antibodies ( lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 ( red ) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green ) and DAPI for nuclear staining ( blue ).

    Techniques Used: Sequencing, In Vitro, Mutagenesis, Incubation, Recombinant, Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Immunofluorescence, Staining

    Effects of Sec16 on RNF183 subcellular localization. (A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 ( green ) and anti-calnexin, GM130, or LAMP1 ( red ) antibodies, and DAPI ( blue ). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC ( top panels) or Sec16A ( middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with ( bottom panels) or without ( top and middle panels) 10 μM MG132 for 12 h.
    Figure Legend Snippet: Effects of Sec16 on RNF183 subcellular localization. (A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 ( green ) and anti-calnexin, GM130, or LAMP1 ( red ) antibodies, and DAPI ( blue ). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC ( top panels) or Sec16A ( middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with ( bottom panels) or without ( top and middle panels) 10 μM MG132 for 12 h.

    Techniques Used: Stable Transfection, Expressing, Transfection, Immunofluorescence, Staining, Inhibition, Incubation

    Effects of Sec16 on other ubiquitin ligases. (A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody ( top panel) and anti-V5 ( second panel) or anti-myc antibodies ( third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p
    Figure Legend Snippet: Effects of Sec16 on other ubiquitin ligases. (A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody ( top panel) and anti-V5 ( second panel) or anti-myc antibodies ( third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p

    Techniques Used: Stable Transfection, Immunoprecipitation, Negative Control, Western Blot, Expressing, Transfection

    Effects of Sec16 on RNF183 protein stability. (A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p
    Figure Legend Snippet: Effects of Sec16 on RNF183 protein stability. (A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot

    28) Product Images from "Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes"

    Article Title: Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458170

    Perturbation of postendocytic trafficking in Rab2 Δ1 garland cells. Garland cells from L3 larvae pulsed for 3 min with TR-avidin (TR-Av) or A488-avidin (A488-Av), followed by a 2 h chase. Single optical sections through the cortical cytoplasm of living garland cells. ( A ) Wild-type cells loaded with TR-avidin and stained with LysoTracker Green (LTG). The lysosomal network is detailed in the high-magnification inset , with three tubular extensions marked by arrowheads and a vacuolar structure by an asterisk . ( B ) Garland cells from control (left) and Rab2 Δ1 (right) animals expressing YFP-Rab7 under direct control of the αTub84BcathD promoter and loaded with TR-avidin. ( C ) Rab2 Δ1 garland cells expressing mCherry-Rab2 and loaded with A488-avidin (live imaging). The inset highlights the tubular network both labeled by endocytic tracer and positive for mCherry-Rab2.
    Figure Legend Snippet: Perturbation of postendocytic trafficking in Rab2 Δ1 garland cells. Garland cells from L3 larvae pulsed for 3 min with TR-avidin (TR-Av) or A488-avidin (A488-Av), followed by a 2 h chase. Single optical sections through the cortical cytoplasm of living garland cells. ( A ) Wild-type cells loaded with TR-avidin and stained with LysoTracker Green (LTG). The lysosomal network is detailed in the high-magnification inset , with three tubular extensions marked by arrowheads and a vacuolar structure by an asterisk . ( B ) Garland cells from control (left) and Rab2 Δ1 (right) animals expressing YFP-Rab7 under direct control of the αTub84BcathD promoter and loaded with TR-avidin. ( C ) Rab2 Δ1 garland cells expressing mCherry-Rab2 and loaded with A488-avidin (live imaging). The inset highlights the tubular network both labeled by endocytic tracer and positive for mCherry-Rab2.

    Techniques Used: Avidin-Biotin Assay, Staining, Expressing, Imaging, Labeling

    29) Product Images from "CHARACTERIZING INTERACTION FORCES BETWEEN ACTIN AND PROTEINS OF THE TROPOMODULIN FAMILY REVEALS THE PRESENCE OF THE N-TERMINAL ACTIN-BINDING SITE IN LEIOMODIN"

    Article Title: CHARACTERIZING INTERACTION FORCES BETWEEN ACTIN AND PROTEINS OF THE TROPOMODULIN FAMILY REVEALS THE PRESENCE OF THE N-TERMINAL ACTIN-BINDING SITE IN LEIOMODIN

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2017.12.005

    Distribution of unbinding forces measured between G-actin and a) Tmod1, b) Tmod3, c) Tmod2, d) Tmod2 1-346 , and e) Tmod2[L73D], respectively. Insets show representative retraction force-curves with specific protein-protein unbinding force peaks. Solid lines show a dynamic peak function model fits to the data presented in the histograms (Origin 9.0, OriginLab Corp., Northampton, MA) ( R 2 > 0.95). Lognormal peak function was used for unimodal distribution whereas Gaussian peak function was used for bimodal distributions. Peak values estimated from dynamic peak function fitting representing the most probable values are given as insets. Tmod2 shows bimodal distribution suggesting that multiple actin binding sites are involved in the interactions to G-actin. Note that peak value of Tmod2 1-346 (64.0±3.6) is similar to the first peak value of Tmod2 (60.3±1.5) (p > 0.05). f) Retraction force curve measured between Tmod1 1-344 [L71D] and G-actin. The interactions between Tmod1 1-344 [L71D] and G-actin were purely repulsive displaying no specific or nonspecific interactions.
    Figure Legend Snippet: Distribution of unbinding forces measured between G-actin and a) Tmod1, b) Tmod3, c) Tmod2, d) Tmod2 1-346 , and e) Tmod2[L73D], respectively. Insets show representative retraction force-curves with specific protein-protein unbinding force peaks. Solid lines show a dynamic peak function model fits to the data presented in the histograms (Origin 9.0, OriginLab Corp., Northampton, MA) ( R 2 > 0.95). Lognormal peak function was used for unimodal distribution whereas Gaussian peak function was used for bimodal distributions. Peak values estimated from dynamic peak function fitting representing the most probable values are given as insets. Tmod2 shows bimodal distribution suggesting that multiple actin binding sites are involved in the interactions to G-actin. Note that peak value of Tmod2 1-346 (64.0±3.6) is similar to the first peak value of Tmod2 (60.3±1.5) (p > 0.05). f) Retraction force curve measured between Tmod1 1-344 [L71D] and G-actin. The interactions between Tmod1 1-344 [L71D] and G-actin were purely repulsive displaying no specific or nonspecific interactions.

    Techniques Used: Binding Assay

    30) Product Images from "Detecting Neurodevelopmental Toxicity of Domoic Acid and Ochratoxin A Using Rat Fetal Neural Stem Cells"

    Article Title: Detecting Neurodevelopmental Toxicity of Domoic Acid and Ochratoxin A Using Rat Fetal Neural Stem Cells

    Journal: Marine Drugs

    doi: 10.3390/md17100566

    Effects of different concentrations of DA and OTA treated for 7 days on cytotoxicity and percentage differentiation of rNSC in relevant directed differentiation medium. Relative cell count was expressed as “total number of cells % control”, and used as an index of cytotoxicity in ( A ) astrocyte, ( C ) neuron, and ( E ) oligodendrocyte differentiation medium, respectively. Percentage differentiation of rNSC into ( B ) astrocytes, ( D ) neurons, and ( F ) oligodendrocytes after 7 days of differentiation process are shown. Differentiated astrocytes, neurons, and oligodendrocytes stained with specific marker GFAP, MAP2, A2B5 or mGalc, respectively. Morphology with neurites in fluorescent, DAPI, and phase contrast overlapping images were used for the quantification. Percentage differentiation = (No. of nuclei of differentiated cells ÷ number of nuclei of total cells) × 100. Data shown are mean ± SE; * p
    Figure Legend Snippet: Effects of different concentrations of DA and OTA treated for 7 days on cytotoxicity and percentage differentiation of rNSC in relevant directed differentiation medium. Relative cell count was expressed as “total number of cells % control”, and used as an index of cytotoxicity in ( A ) astrocyte, ( C ) neuron, and ( E ) oligodendrocyte differentiation medium, respectively. Percentage differentiation of rNSC into ( B ) astrocytes, ( D ) neurons, and ( F ) oligodendrocytes after 7 days of differentiation process are shown. Differentiated astrocytes, neurons, and oligodendrocytes stained with specific marker GFAP, MAP2, A2B5 or mGalc, respectively. Morphology with neurites in fluorescent, DAPI, and phase contrast overlapping images were used for the quantification. Percentage differentiation = (No. of nuclei of differentiated cells ÷ number of nuclei of total cells) × 100. Data shown are mean ± SE; * p

    Techniques Used: Cell Counting, Staining, Marker

    Effects of different concentrations of DA on the differentiation of rNSC into oligodendrocytes. rNSC cultured in oligodendrocyte differentiation media with and without DA for 7 days. Mature oligodendrocytes were stained with specific marker A2B5 and nucleus marker DAPI. Representative fluorescent images of oligodendrocyte differentiation ( A ) without DA (control), ( B ) with 0.05 μM of DA, and ( C ) with 5 μM of DA are depicted. Scale bar indicates 200 μm at 20× magnification.
    Figure Legend Snippet: Effects of different concentrations of DA on the differentiation of rNSC into oligodendrocytes. rNSC cultured in oligodendrocyte differentiation media with and without DA for 7 days. Mature oligodendrocytes were stained with specific marker A2B5 and nucleus marker DAPI. Representative fluorescent images of oligodendrocyte differentiation ( A ) without DA (control), ( B ) with 0.05 μM of DA, and ( C ) with 5 μM of DA are depicted. Scale bar indicates 200 μm at 20× magnification.

    Techniques Used: Cell Culture, Staining, Marker

    rNSC neurospheres cultured in complete StemPro NSC SFM medium without differentiation factors, or with oligodendrocyte differentiation medium. ( A ) Neurospheres cultured in slide chambers coated with Geltrex showed fusing neurospheres. ( B ) Neurospheres cultured as a suspension in uncoated slide chambers showed fused and fusing neurospheres that were stained with NSC marker nestin. ( C ) Neurospheres cultured in oligodendrocyte directed differentiation medium on day 2, and ( D ) on day 4 of the experiment, showing immature oligodendrocytes coming out from the neurospheres in phase contrast images. ( E ) Day 9 fluorescent image, after staining with oligodendrocyte-specific marker A2B5 (green), showing immature oligodendrocytes coming out from the neurospheres, and nucleus marker DAPI (blue) confirm fully differentiated oligodendrocytes. Scale bar indicates 200 μm or 400 μm at 20× magnification.
    Figure Legend Snippet: rNSC neurospheres cultured in complete StemPro NSC SFM medium without differentiation factors, or with oligodendrocyte differentiation medium. ( A ) Neurospheres cultured in slide chambers coated with Geltrex showed fusing neurospheres. ( B ) Neurospheres cultured as a suspension in uncoated slide chambers showed fused and fusing neurospheres that were stained with NSC marker nestin. ( C ) Neurospheres cultured in oligodendrocyte directed differentiation medium on day 2, and ( D ) on day 4 of the experiment, showing immature oligodendrocytes coming out from the neurospheres in phase contrast images. ( E ) Day 9 fluorescent image, after staining with oligodendrocyte-specific marker A2B5 (green), showing immature oligodendrocytes coming out from the neurospheres, and nucleus marker DAPI (blue) confirm fully differentiated oligodendrocytes. Scale bar indicates 200 μm or 400 μm at 20× magnification.

    Techniques Used: Cell Culture, Staining, Marker

    Undifferentiated control rat neural stem cells (rNSC) and rNSC differentiated into oligodendrocytes, astrocytes, and neurons after 7 days in their respective differentiation process. ( A ) Control rNSC cultured in rat neural stem cell culture medium, then immune-stained with neural stem cell-specific marker Nestin (green). ( B ) rNSC cultured in oligodendrocyte differentiation medium, immune-stained with oligodendrocyte-specific markers A2B5 (green). ( C ) Oligodendrocytes immuno-stained with oligodendrocyte-specific marker Galactocerebroside (pink). ( D ) rNSC cultured in astrocyte directed differentiation medium and then immune-stained with astrocyte-specific marker GFAP (green). ( E ) Phase contrast-fluorescent overlapped image of mature oligodendrocytes. ( F ) rNSC cultured in neuron directed differentiation medium, immuno-stained with neuron-specific marker MAP2 clone AP18 (pink). Nucleus marker DAPI (blue). Scale bar indicates 200 μm at 20× magnification.
    Figure Legend Snippet: Undifferentiated control rat neural stem cells (rNSC) and rNSC differentiated into oligodendrocytes, astrocytes, and neurons after 7 days in their respective differentiation process. ( A ) Control rNSC cultured in rat neural stem cell culture medium, then immune-stained with neural stem cell-specific marker Nestin (green). ( B ) rNSC cultured in oligodendrocyte differentiation medium, immune-stained with oligodendrocyte-specific markers A2B5 (green). ( C ) Oligodendrocytes immuno-stained with oligodendrocyte-specific marker Galactocerebroside (pink). ( D ) rNSC cultured in astrocyte directed differentiation medium and then immune-stained with astrocyte-specific marker GFAP (green). ( E ) Phase contrast-fluorescent overlapped image of mature oligodendrocytes. ( F ) rNSC cultured in neuron directed differentiation medium, immuno-stained with neuron-specific marker MAP2 clone AP18 (pink). Nucleus marker DAPI (blue). Scale bar indicates 200 μm at 20× magnification.

    Techniques Used: Cell Culture, Stem Cell Culture, Staining, Marker

    31) Product Images from "Multiple cytosolic DNA sensors bind plasmid DNA after transfection"

    Article Title: Multiple cytosolic DNA sensors bind plasmid DNA after transfection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz768

    Effects of pDNA transfection on IFN β production and expression of putative cytosolic DNA sensors. ( A ) IFN β mRNA levels 4 h and protein levels 6 h after transfection using electroporation with gWizBlank normalized to control group ( n = 3–10). ( B ) IFN β mRNA levels 4 h after transfection using Transit (T) normalized to control group (C) ( n = 6–8). ( C ) DDX60, DAI/ZBP1 and p204 mRNA levels 4 h after electroporation normalized to control group ( n = 3–9). ( D ) DDX60, DAI/ZBP1 and p204 mRNA levels 4 h after chemical transfection (T) normalized to control group (C) ( n = 8). ( E ) Representative western blots of DDX60, DAI/ZBP1 and p204 proteins 8 h after transfection using EP2. ( F ) DDX60, DAI/ZBP1 and p204 upregulation 8 h after transfection using EP2 normalized to control group ( n = 3 for each protein). * P
    Figure Legend Snippet: Effects of pDNA transfection on IFN β production and expression of putative cytosolic DNA sensors. ( A ) IFN β mRNA levels 4 h and protein levels 6 h after transfection using electroporation with gWizBlank normalized to control group ( n = 3–10). ( B ) IFN β mRNA levels 4 h after transfection using Transit (T) normalized to control group (C) ( n = 6–8). ( C ) DDX60, DAI/ZBP1 and p204 mRNA levels 4 h after electroporation normalized to control group ( n = 3–9). ( D ) DDX60, DAI/ZBP1 and p204 mRNA levels 4 h after chemical transfection (T) normalized to control group (C) ( n = 8). ( E ) Representative western blots of DDX60, DAI/ZBP1 and p204 proteins 8 h after transfection using EP2. ( F ) DDX60, DAI/ZBP1 and p204 upregulation 8 h after transfection using EP2 normalized to control group ( n = 3 for each protein). * P

    Techniques Used: Transfection, Expressing, Electroporation, Western Blot

    Protein immunoprecipitation and pDNA pull-down. ( A ) Schematic representation of biotinylated pDNA pull-down. ( B ) Representative membranes demonstrating proteins’ binding to pDNA detected by western blotting. Whole-cell C2C12 lysate serves as positive control (bottom line). ( C ) Time course of CREB, DAI/ZBP1, DHX9 and p204 binding to pDNA ( n = 3 for each protein). All time points represent time after gWizGFP transfection. ( D ) Schematic representation of protein immunoprecipitation. ( E ) gWizGFP plasmid copy number normalized to CD4 group ( n = 3 for CREB, DHX9 and cGAS groups, n = 6 for the other groups). No statistical difference was observed within any group whether the CMV promoter (blue) or the GFP open reading frame (green) was targeted. * P
    Figure Legend Snippet: Protein immunoprecipitation and pDNA pull-down. ( A ) Schematic representation of biotinylated pDNA pull-down. ( B ) Representative membranes demonstrating proteins’ binding to pDNA detected by western blotting. Whole-cell C2C12 lysate serves as positive control (bottom line). ( C ) Time course of CREB, DAI/ZBP1, DHX9 and p204 binding to pDNA ( n = 3 for each protein). All time points represent time after gWizGFP transfection. ( D ) Schematic representation of protein immunoprecipitation. ( E ) gWizGFP plasmid copy number normalized to CD4 group ( n = 3 for CREB, DHX9 and cGAS groups, n = 6 for the other groups). No statistical difference was observed within any group whether the CMV promoter (blue) or the GFP open reading frame (green) was targeted. * P

    Techniques Used: Immunoprecipitation, Binding Assay, Western Blot, Positive Control, Transfection, Plasmid Preparation

    Kinetics of mRNA expression. ( A ) IFN β, ( B ) GFP, ( C ) DAI/ZBP1, DDX60, and p204 mRNA levels at 0, 0.5, 1, 5 and 24 h after transfection ( n = 5–8). * P
    Figure Legend Snippet: Kinetics of mRNA expression. ( A ) IFN β, ( B ) GFP, ( C ) DAI/ZBP1, DDX60, and p204 mRNA levels at 0, 0.5, 1, 5 and 24 h after transfection ( n = 5–8). * P

    Techniques Used: Expressing, Transfection

    Effect of DAIZBP1, p204 and DHX9 silencing on the mRNA levels of IFN β, STING and cGAS. Levels of mRNA of cytosolic DNA sensors (DAI/ZBP1, p204 and DHX9), IFN β, STING and cGAS 4 h after transfection with pDNA in cells treated for 48 h with siRNAs as noted. Levels of mRNA were normalised to the mRNA levels in control cells treated with scrambled siRNA ( n = 3 for p204 and n = 4 for DAI/ZBP1 and DHX9). *** P
    Figure Legend Snippet: Effect of DAIZBP1, p204 and DHX9 silencing on the mRNA levels of IFN β, STING and cGAS. Levels of mRNA of cytosolic DNA sensors (DAI/ZBP1, p204 and DHX9), IFN β, STING and cGAS 4 h after transfection with pDNA in cells treated for 48 h with siRNAs as noted. Levels of mRNA were normalised to the mRNA levels in control cells treated with scrambled siRNA ( n = 3 for p204 and n = 4 for DAI/ZBP1 and DHX9). *** P

    Techniques Used: Transfection

    32) Product Images from "Microwaves from GSM Mobile Telephones Affect 53BP1 and ?-H2AX Foci in Human Lymphocytes from Hypersensitive and Healthy Persons"

    Article Title: Microwaves from GSM Mobile Telephones Affect 53BP1 and ?-H2AX Foci in Human Lymphocytes from Hypersensitive and Healthy Persons

    Journal: Environmental Health Perspectives

    doi: 10.1289/ehp.7561

    Images of fixed human lymphocytes (counterstained blue with ToPro-3-iodide) showing 53BP1 foci (stained green with Alexa fluor 488) and γ-H2AX foci (stained red with Cy3) as revealed by immunostaining and confocal laser microscopy of cells from subject 501. Significantly fewer foci were observed after 1 hr exposure to 915 MHz and heat shock (41°C) than in control cells. Exposure to 905 MHz resulted in a statistically significant increase in the number of 53BP1 foci in cells from this subject ( Table 3 ). Bar = 10 μm.
    Figure Legend Snippet: Images of fixed human lymphocytes (counterstained blue with ToPro-3-iodide) showing 53BP1 foci (stained green with Alexa fluor 488) and γ-H2AX foci (stained red with Cy3) as revealed by immunostaining and confocal laser microscopy of cells from subject 501. Significantly fewer foci were observed after 1 hr exposure to 915 MHz and heat shock (41°C) than in control cells. Exposure to 905 MHz resulted in a statistically significant increase in the number of 53BP1 foci in cells from this subject ( Table 3 ). Bar = 10 μm.

    Techniques Used: Staining, Immunostaining, Microscopy

    33) Product Images from "Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes"

    Article Title: Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458170

    Lysosomal traffic and autophagy are perturbed by loss of Rab2. ( A ) Localization of ubiquitously expressed GFP-LAMP in relation to LysoTracker Red staining in L3 salivary glands. Single optical sections of gland cortex near the basolateral surface (live imaging). Arrowheads indicate GFP-LAMP-positive vesicular structures not labeled with LysoTracker Red. Arrows indicate structures labeled with both GFP-LAMP and LysoTracker Red. Brackets indicate coclustering of GFP-LAMP-positive structures with structures labeled with LysoTracker Red. ( B ) Quantification of data in (E), from 9 control, 14 Rab2 Δ1 and 10 lt 11 /Df larvae. The fraction of GFP-LAMP signal located outside of LysoTracker Red-positive structures is shown. ( C ) Localization of GFP-LAMP relative to LysoTracker Red in the fat body. Left , late L3 fat bodies from control, Rab2 Δ1 and lt 11 /Df larvae ubiquitously expressing GFP-LAMP. Single optical sections (live imaging). For better visibility, the intensity of the LysoTracker Red signal in the Rab2 Δ1 and lt 11 /Df fat body was digitally increased relative to control. Right , intensity scatter plots for GFP-LAMP versus LysoTracker Red. Warm colors mark a high point density in the plot. The Pearson correlation coefficient (PCC) is indicated. ( D and E ) Quantification of ( C ). Control and Rab2 Δ1 each represented by 5 larvae, lt 11 /Df by 6 larvae. ( D ) Correlation between the GFP-LAMP and LysoTracker Red signals. ( E ) Mean LysoTracker Red signal density ( left ) and cross-sectional area of LysoTracker Red-positive structures ( right ). ( F ) Eye imaginal discs of control and Rab2 Δ1 late L3 larvae expressing the GFP-mCherry-Atg8a autophagy reporter. Arrows indicate accumulation of GFP and mCherry dual-positive autophagosomes in the ommatidia of Rab2 Δ1 imaginal discs. Live confocal imaging of eye disc area posterior to the morphogenetic furrow. ( G ) Quantification of ( F ). The GFP:mCherry signal ratio in mCherry-positive autophagosomal structures in 8 w 1118 and 7 Rab2 Δ1 imaginal discs is shown. ( H ) Eye pigmentation is not perturbed by loss of Rab2 . Micrographs of the eyes of control and Rab2 Δ1 pharate adult flies. ( B, D , and E ) ANOVA followed by Tukey HSD test, ( G ) Unpaired Student’s t test.
    Figure Legend Snippet: Lysosomal traffic and autophagy are perturbed by loss of Rab2. ( A ) Localization of ubiquitously expressed GFP-LAMP in relation to LysoTracker Red staining in L3 salivary glands. Single optical sections of gland cortex near the basolateral surface (live imaging). Arrowheads indicate GFP-LAMP-positive vesicular structures not labeled with LysoTracker Red. Arrows indicate structures labeled with both GFP-LAMP and LysoTracker Red. Brackets indicate coclustering of GFP-LAMP-positive structures with structures labeled with LysoTracker Red. ( B ) Quantification of data in (E), from 9 control, 14 Rab2 Δ1 and 10 lt 11 /Df larvae. The fraction of GFP-LAMP signal located outside of LysoTracker Red-positive structures is shown. ( C ) Localization of GFP-LAMP relative to LysoTracker Red in the fat body. Left , late L3 fat bodies from control, Rab2 Δ1 and lt 11 /Df larvae ubiquitously expressing GFP-LAMP. Single optical sections (live imaging). For better visibility, the intensity of the LysoTracker Red signal in the Rab2 Δ1 and lt 11 /Df fat body was digitally increased relative to control. Right , intensity scatter plots for GFP-LAMP versus LysoTracker Red. Warm colors mark a high point density in the plot. The Pearson correlation coefficient (PCC) is indicated. ( D and E ) Quantification of ( C ). Control and Rab2 Δ1 each represented by 5 larvae, lt 11 /Df by 6 larvae. ( D ) Correlation between the GFP-LAMP and LysoTracker Red signals. ( E ) Mean LysoTracker Red signal density ( left ) and cross-sectional area of LysoTracker Red-positive structures ( right ). ( F ) Eye imaginal discs of control and Rab2 Δ1 late L3 larvae expressing the GFP-mCherry-Atg8a autophagy reporter. Arrows indicate accumulation of GFP and mCherry dual-positive autophagosomes in the ommatidia of Rab2 Δ1 imaginal discs. Live confocal imaging of eye disc area posterior to the morphogenetic furrow. ( G ) Quantification of ( F ). The GFP:mCherry signal ratio in mCherry-positive autophagosomal structures in 8 w 1118 and 7 Rab2 Δ1 imaginal discs is shown. ( H ) Eye pigmentation is not perturbed by loss of Rab2 . Micrographs of the eyes of control and Rab2 Δ1 pharate adult flies. ( B, D , and E ) ANOVA followed by Tukey HSD test, ( G ) Unpaired Student’s t test.

    Techniques Used: Staining, Imaging, Labeling, Expressing, Periodic Counter-current Chromatography

    Perturbed biosynthetic delivery of LAMP to LEs in Rab2 and HOPS but not Gie / Arl8 mutant garland cells. ( A ) Garland cells from L3 larvae of the indicated genotypes expressing GFP-LAMP under direct control of the αTub84B promoter. Single optical sections through the cortical cytoplasm (live imaging). The cells were pulsed with TR-avidin (3 min), then chased for 2 h. Insets depict GFP-LAMP signal accumulating in tracer-negative vesicular structures ( white arrows ) and perivacuolar rings ( white closed and open arrowheads ) around tracer-positive vacuoles in Rab2 Δ1 , HOPS mutants and Rab2 Δ1 ; Vps39 Δ1 double mutants, or in the lumen ( blue arrowheads ) of tracer-positive and tracer-negative vacuoles in Gie e00336 hemizygotes. Blue arrow indicates a vacuole with some degree of lumenal GFP accumulation in a Vps39 Δ1 /Df cell. ( B and C ) Quantification of data in ( A ). ( B ) Density of the GFP-LAMP signal inside tracer-filled vacuoles relative to the density in a 0.2 µm wide external band surrounding the vacuoles. ( C ) Density of high-intensity GFP-LAMP vesicles larger than 0.1 µm 2 (equivalent to a diameter of ~ 360 nm) and located within a 2-µm band surrounding the tracer-filled vacuoles. In ( B and C ), the following numbers of garland cell clusters were analyzed: 10 Rab2 Δ1 , 16 Vps39 Δ1 /Df , 19 lt 22 /Df , 10 Rab2 Δ1 ; Vps39 Δ1 , and 11 Gie e00336 /Df . ( B ) P values in black and blue represent comparisons to Rab2 Δ1 ; Vps39 Δ1 and Gie e00336 /Df , respectively. ( C ) Thick horizontal lines indicate pooling of genotypes before statistical testing. ANOVA followed by Tukey HSD test ( B ) and Scheffé contrasts ( C ).
    Figure Legend Snippet: Perturbed biosynthetic delivery of LAMP to LEs in Rab2 and HOPS but not Gie / Arl8 mutant garland cells. ( A ) Garland cells from L3 larvae of the indicated genotypes expressing GFP-LAMP under direct control of the αTub84B promoter. Single optical sections through the cortical cytoplasm (live imaging). The cells were pulsed with TR-avidin (3 min), then chased for 2 h. Insets depict GFP-LAMP signal accumulating in tracer-negative vesicular structures ( white arrows ) and perivacuolar rings ( white closed and open arrowheads ) around tracer-positive vacuoles in Rab2 Δ1 , HOPS mutants and Rab2 Δ1 ; Vps39 Δ1 double mutants, or in the lumen ( blue arrowheads ) of tracer-positive and tracer-negative vacuoles in Gie e00336 hemizygotes. Blue arrow indicates a vacuole with some degree of lumenal GFP accumulation in a Vps39 Δ1 /Df cell. ( B and C ) Quantification of data in ( A ). ( B ) Density of the GFP-LAMP signal inside tracer-filled vacuoles relative to the density in a 0.2 µm wide external band surrounding the vacuoles. ( C ) Density of high-intensity GFP-LAMP vesicles larger than 0.1 µm 2 (equivalent to a diameter of ~ 360 nm) and located within a 2-µm band surrounding the tracer-filled vacuoles. In ( B and C ), the following numbers of garland cell clusters were analyzed: 10 Rab2 Δ1 , 16 Vps39 Δ1 /Df , 19 lt 22 /Df , 10 Rab2 Δ1 ; Vps39 Δ1 , and 11 Gie e00336 /Df . ( B ) P values in black and blue represent comparisons to Rab2 Δ1 ; Vps39 Δ1 and Gie e00336 /Df , respectively. ( C ) Thick horizontal lines indicate pooling of genotypes before statistical testing. ANOVA followed by Tukey HSD test ( B ) and Scheffé contrasts ( C ).

    Techniques Used: Mutagenesis, Expressing, Imaging, Avidin-Biotin Assay

    Rab2 is present on the limiting membranes of LEs and lysosomes. ( A to E ) High-magnification single optical sections (live imaging) through cortical garland cells ( A to C ) or cortical salivary gland cells from L3 larvae ( D and E ). ( A ) Garland cells from larva overexpressing wild-type pHluorin-Rab2 ( top ) or gain-of-function pHluorin-Rab2 Q65L ( bottom ) pulsed with TR-avidin (3 min) and chased for 2 h. ( B ) Garland cells from larva expressing GFP-Rab2 under endogenous promoter regulation, incubated for 7ʹ with TR-avidin ( top ), or pulsed for 3 min with TR-avidin and chased for 2 h ( bottom ). ( C ) Garland cell from larva overexpressing gain-of-function mCherry-Rab2 Q65L and expressing GFP-LAMP under control of the αTub84B promoter, pulsed with A647-BSA (3 min) and chased for 2 h. ( D ) Salivary gland cells from larva expressing GFP-Rab2 under endogenous promotor regulation, stained with LysoTracker Red (LTR). GFP-Rab2 labels LTR-positive lysosomal tubules ( top, open arrowheads ) and membranes of LTR-positive lysosomal vacuoles ( bottom, closed arrowheads ). Inset shows a pair of adjacent LTR and GFP-Rab2-positive vacuoles at higher magnification. The strongly GFP-Rab2-labeled, LTR-negative structures are mostly Golgi bodies ( arrows ). ( E ) Salivary gland cells from larva overexpressing gain-of-function pHluorin-Rab2 Q65L , stained with LTR. Top , Tangential section of the basolateral gland surface. The limiting membranes of smaller LTR-positive lysosomal vacuoles ( arrows ) and lysosomal tubules extending from them ( open arrowheads ) are labeled with pHluorin-Rab2 Q65L . Bottom , Giant lysosomal vacuole containing strongly LTR-labeled internal vesicles. The limiting membrane is labeled with pHluorin-Rab2 Q65L ( arrowheads ). Plane of section deeper within the gland cytoplasm compared to top.
    Figure Legend Snippet: Rab2 is present on the limiting membranes of LEs and lysosomes. ( A to E ) High-magnification single optical sections (live imaging) through cortical garland cells ( A to C ) or cortical salivary gland cells from L3 larvae ( D and E ). ( A ) Garland cells from larva overexpressing wild-type pHluorin-Rab2 ( top ) or gain-of-function pHluorin-Rab2 Q65L ( bottom ) pulsed with TR-avidin (3 min) and chased for 2 h. ( B ) Garland cells from larva expressing GFP-Rab2 under endogenous promoter regulation, incubated for 7ʹ with TR-avidin ( top ), or pulsed for 3 min with TR-avidin and chased for 2 h ( bottom ). ( C ) Garland cell from larva overexpressing gain-of-function mCherry-Rab2 Q65L and expressing GFP-LAMP under control of the αTub84B promoter, pulsed with A647-BSA (3 min) and chased for 2 h. ( D ) Salivary gland cells from larva expressing GFP-Rab2 under endogenous promotor regulation, stained with LysoTracker Red (LTR). GFP-Rab2 labels LTR-positive lysosomal tubules ( top, open arrowheads ) and membranes of LTR-positive lysosomal vacuoles ( bottom, closed arrowheads ). Inset shows a pair of adjacent LTR and GFP-Rab2-positive vacuoles at higher magnification. The strongly GFP-Rab2-labeled, LTR-negative structures are mostly Golgi bodies ( arrows ). ( E ) Salivary gland cells from larva overexpressing gain-of-function pHluorin-Rab2 Q65L , stained with LTR. Top , Tangential section of the basolateral gland surface. The limiting membranes of smaller LTR-positive lysosomal vacuoles ( arrows ) and lysosomal tubules extending from them ( open arrowheads ) are labeled with pHluorin-Rab2 Q65L . Bottom , Giant lysosomal vacuole containing strongly LTR-labeled internal vesicles. The limiting membrane is labeled with pHluorin-Rab2 Q65L ( arrowheads ). Plane of section deeper within the gland cytoplasm compared to top.

    Techniques Used: Imaging, Avidin-Biotin Assay, Expressing, Incubation, Staining, Labeling

    Accumulation of GFP-LAMP in the CNS of Rab2 , HOPS and Gie / Arl8 mutant larvae. ( A ) Micrographs of right brain hemispheres from fixed L3 larvae ubiquitously expressing GFP-LAMP. The plane of section for the top row (labeled ‘1’) is near the dorsal surface of the hemisphere, while that for the middle row (labeled ‘2’) is about 2 µm deeper in the preparation. The schematic in ( B ) shows the approximate position of the two optical sections in a live brain, with labels ‘1’ and ‘2’ corresponding to those in ( A ). ( A , middle ) A prominent structure ( brackets ) with clusters of GFP-LAMP granules appeared exclusively in Rab2 Δ1 mutants and Rab2 Δ1 ; Vps39 Δ1 double mutants. ( A , bottom ) Areas representative of GFP-LAMP signal accumulation at high resolution. Large GFP-LAMP-positive vacuoles, only occurring in Rab2 Δ1 ; Vps39 Δ1 double mutants, are also shown ( bottom inset ). Single optical slices; medial is right , anterior down . ( C ) The amount of GFP signal contained in GFP-LAMP granules in the dorsal aspect of the brain hemispheres. The following numbers of larvae (hemispheres) were analyzed (ANOVA followed by Tukey HSD test): 16 w 1118 , 15 Rab2 Δ1 , 7 Vps39 Δ1 /Df , 10 lt 22 /Df , 9 Rab2 Δ1 ; Vps39 Δ1 , and 11 Gie e00336 /Df. P -values in black and blue represent comparisons to w 1118 and Rab2 Δ1 ; Vps39 Δ1 , respectively.
    Figure Legend Snippet: Accumulation of GFP-LAMP in the CNS of Rab2 , HOPS and Gie / Arl8 mutant larvae. ( A ) Micrographs of right brain hemispheres from fixed L3 larvae ubiquitously expressing GFP-LAMP. The plane of section for the top row (labeled ‘1’) is near the dorsal surface of the hemisphere, while that for the middle row (labeled ‘2’) is about 2 µm deeper in the preparation. The schematic in ( B ) shows the approximate position of the two optical sections in a live brain, with labels ‘1’ and ‘2’ corresponding to those in ( A ). ( A , middle ) A prominent structure ( brackets ) with clusters of GFP-LAMP granules appeared exclusively in Rab2 Δ1 mutants and Rab2 Δ1 ; Vps39 Δ1 double mutants. ( A , bottom ) Areas representative of GFP-LAMP signal accumulation at high resolution. Large GFP-LAMP-positive vacuoles, only occurring in Rab2 Δ1 ; Vps39 Δ1 double mutants, are also shown ( bottom inset ). Single optical slices; medial is right , anterior down . ( C ) The amount of GFP signal contained in GFP-LAMP granules in the dorsal aspect of the brain hemispheres. The following numbers of larvae (hemispheres) were analyzed (ANOVA followed by Tukey HSD test): 16 w 1118 , 15 Rab2 Δ1 , 7 Vps39 Δ1 /Df , 10 lt 22 /Df , 9 Rab2 Δ1 ; Vps39 Δ1 , and 11 Gie e00336 /Df. P -values in black and blue represent comparisons to w 1118 and Rab2 Δ1 ; Vps39 Δ1 , respectively.

    Techniques Used: Mutagenesis, Expressing, Labeling

    Rab2 genetics, expression pattern and loss-of-function phenotype. ( A ) Map of the Rab2 genomic region, with neighboring genes and 2 large deficiencies covering Rab2 . The region covered by the HA-Rab2 transgene and the structure of the Rab2 locus is indicated, as well as the location of the HA and GFP tags inserted in the genomic transgenes, the P -element used in excision mutagenesis, and the extent of the deletion in Rab2 Δ1 . Coding region in red . ( B ) Developmental survival profile. The initial numbers of flies were: w 1118 , 90; Rab2 Δ1 /+ , 92; Rab2 Δ1 , 92; Rab2 Δ1 /Df(2R)BSC326 , 89; Rab2 Δ1 /Df(2R)BSC326;tub-Gal4,mCherry-Rab2/+ , 32.( C ) Confocal sum intensity projections showing the expression of HA-Rab2 in filleted L3 larvae carrying two copies of the HA-Rab2 allele in a Rab2 Δ1 background, and w 1118 controls without HA-Rab2 . Anti-HA staining was supplemented by anti-HRP staining to mark the nervous system. Br, brain hemispheres; VNC, ventral nerve cord; PN, peripheral nerves, M, muscles; RG, ring gland; SG, salivary gland. ( D and E ) Accumulation of GFP-LAMP in the CNS of Rab2 Δ1 mutant larvae. ( D ) Maximum projections of fixed CNS from control, Rab2 Δ1 and rescued L3 larvae ubiquitously expressing GFP-LAMP. VNC, ventral nerve cord; LH/RH, left/right hemisphere. ( E ) Amount of LAMP signal in GFP-LAMP granules in the VNC and hemispheres. Each genotype represented by 5 CNS preparations. ANOVAs followed by the Tukey honest significant difference (HSD) test. ( F and G ) Localization of GFP-LAMP-containing granules accumulating in Rab2 Δ1 L3 brains, in relation to the Golgi and late endosomes. High-magnification single optical sections through the surface of a Rab2 Δ1 brain hemisphere prepared as in ( D ) and stained for GM130 to mark Golgi bodies ( F ) or Rab7 to mark late endosomes ( G ). Arrows indicate accumulations of GFP-LAMP coinciding with Rab7-positive LEs. Arrowheads indicate Rab7-negative GFP-LAMP granules. The detailed genotypes included in this and the following figures, supplemental figures, and videos are listed in Table S1.
    Figure Legend Snippet: Rab2 genetics, expression pattern and loss-of-function phenotype. ( A ) Map of the Rab2 genomic region, with neighboring genes and 2 large deficiencies covering Rab2 . The region covered by the HA-Rab2 transgene and the structure of the Rab2 locus is indicated, as well as the location of the HA and GFP tags inserted in the genomic transgenes, the P -element used in excision mutagenesis, and the extent of the deletion in Rab2 Δ1 . Coding region in red . ( B ) Developmental survival profile. The initial numbers of flies were: w 1118 , 90; Rab2 Δ1 /+ , 92; Rab2 Δ1 , 92; Rab2 Δ1 /Df(2R)BSC326 , 89; Rab2 Δ1 /Df(2R)BSC326;tub-Gal4,mCherry-Rab2/+ , 32.( C ) Confocal sum intensity projections showing the expression of HA-Rab2 in filleted L3 larvae carrying two copies of the HA-Rab2 allele in a Rab2 Δ1 background, and w 1118 controls without HA-Rab2 . Anti-HA staining was supplemented by anti-HRP staining to mark the nervous system. Br, brain hemispheres; VNC, ventral nerve cord; PN, peripheral nerves, M, muscles; RG, ring gland; SG, salivary gland. ( D and E ) Accumulation of GFP-LAMP in the CNS of Rab2 Δ1 mutant larvae. ( D ) Maximum projections of fixed CNS from control, Rab2 Δ1 and rescued L3 larvae ubiquitously expressing GFP-LAMP. VNC, ventral nerve cord; LH/RH, left/right hemisphere. ( E ) Amount of LAMP signal in GFP-LAMP granules in the VNC and hemispheres. Each genotype represented by 5 CNS preparations. ANOVAs followed by the Tukey honest significant difference (HSD) test. ( F and G ) Localization of GFP-LAMP-containing granules accumulating in Rab2 Δ1 L3 brains, in relation to the Golgi and late endosomes. High-magnification single optical sections through the surface of a Rab2 Δ1 brain hemisphere prepared as in ( D ) and stained for GM130 to mark Golgi bodies ( F ) or Rab7 to mark late endosomes ( G ). Arrows indicate accumulations of GFP-LAMP coinciding with Rab7-positive LEs. Arrowheads indicate Rab7-negative GFP-LAMP granules. The detailed genotypes included in this and the following figures, supplemental figures, and videos are listed in Table S1.

    Techniques Used: Expressing, Mutagenesis, Staining

    34) Product Images from "Potassium channel blocker, 4-Aminopyridine-3-Methanol, restores axonal conduction in spinal cord of an animal model of multiple sclerosis"

    Article Title: Potassium channel blocker, 4-Aminopyridine-3-Methanol, restores axonal conduction in spinal cord of an animal model of multiple sclerosis

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2010.11.004

    Images of thoracic cross sections of spinal cords from control (A-C) and EAE (D-F) mice labeled with fluoromyelin (red) and anti-NF200 (green). No noticeable demyelinated areas were found in the white matter of control mice spinal cord (A – C). In EAE mouse spinal cord sections, a typical demyelinated region in the white matter is shown (arrow) (D - F). G. A photomicrograph displays the higher magnification of the region circumscribed in F. Note that many axons are labeled without corresponding myelin staining. The open arrow indicates area where axons lack of myelin and filled arrow denotes area of axons with normal myelin appearance. Asterisk indicates gray matter of spinal cord. Scale bar = 500 µm (A - F); 200µm (G).
    Figure Legend Snippet: Images of thoracic cross sections of spinal cords from control (A-C) and EAE (D-F) mice labeled with fluoromyelin (red) and anti-NF200 (green). No noticeable demyelinated areas were found in the white matter of control mice spinal cord (A – C). In EAE mouse spinal cord sections, a typical demyelinated region in the white matter is shown (arrow) (D - F). G. A photomicrograph displays the higher magnification of the region circumscribed in F. Note that many axons are labeled without corresponding myelin staining. The open arrow indicates area where axons lack of myelin and filled arrow denotes area of axons with normal myelin appearance. Asterisk indicates gray matter of spinal cord. Scale bar = 500 µm (A - F); 200µm (G).

    Techniques Used: Mouse Assay, Labeling, Staining

    35) Product Images from "Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei"

    Article Title: Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.211730

    MiRF172 and TAC102 after p197 RNAi depletion and recovery after removal of tet in γL262P p197 RNAi BSF T. brucei cells. (A) Colocalization of MiRF172–PTP with TAC102 in γL262P p197 RNAi BSF cells. Localization of MiRF172–PTP (magenta) and TAC102 (green) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of γL262P p197 RNAi BSF T. brucei cells. MiRF172–PTP was detected with anti-Protein A antibody. TAC102 was detected with anti-TAC102 monoclonal mouse antibody. The kDNA and the nucleus were stained with DAPI (cyan). The inset shows a higher magnification view. (B) TAC recovery experiment in γL262P p197 RNAi BSF T. brucei cells. To detect MiRF172–PTP, TAC102 and DNA the same antibodies and reagents as in A were used. The pictures were obtained under the same conditions as in A. The basal bodies (red) were detected with the YL1/2 monoclonal antibody. - tet, uninduced cells; d5 post induction, MiRF172-depleted cells at day 5 of RNAi (RNAi was induced by addition of tet); d4 post recovery, after 5 days of RNAi, tet was removed and cells were grown for 4 additional days. (C) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P p197 RNAi induced and uninduced cells ( n ≥113 for each time point). K, kDNA; N, nucleus. (D) Quantitative analysis of TAC102 in γL262P p197 RNAi cells without tet (no tet), with tet at day five (d5 p.i.) as well as 2 days after removal of tet (post recovery; d2 p.r.) and at day 4 post recovery (d4 p.r.) ( n ≥105 for each time point). (E) Quantitative analysis of the MiRF172–PTP signal in in γL262P p197 RNAi cells as in D ( n ≥105 for each time point). (F) Western blot analysis of γL262P p197 RNAi BSF cells. Total protein isolated from uninduced cells (−tet), cells induced with tet for 5 days (d5 p.i.) and cells released from p197 RNAi at day 2 (d2 p.r.) and day 4 post recovery (d4 p.r.) was used. C-terminally PTP-tagged MiRF172 was detected with the anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. EF1α serves as a loading control. Arrowheads point to the TAC102 and MiRF172 signals. PH, phase contrast. Scale bars: 5 µm.
    Figure Legend Snippet: MiRF172 and TAC102 after p197 RNAi depletion and recovery after removal of tet in γL262P p197 RNAi BSF T. brucei cells. (A) Colocalization of MiRF172–PTP with TAC102 in γL262P p197 RNAi BSF cells. Localization of MiRF172–PTP (magenta) and TAC102 (green) is represented by maximum intensity projections from immunofluorescence microscopy image stacks of γL262P p197 RNAi BSF T. brucei cells. MiRF172–PTP was detected with anti-Protein A antibody. TAC102 was detected with anti-TAC102 monoclonal mouse antibody. The kDNA and the nucleus were stained with DAPI (cyan). The inset shows a higher magnification view. (B) TAC recovery experiment in γL262P p197 RNAi BSF T. brucei cells. To detect MiRF172–PTP, TAC102 and DNA the same antibodies and reagents as in A were used. The pictures were obtained under the same conditions as in A. The basal bodies (red) were detected with the YL1/2 monoclonal antibody. - tet, uninduced cells; d5 post induction, MiRF172-depleted cells at day 5 of RNAi (RNAi was induced by addition of tet); d4 post recovery, after 5 days of RNAi, tet was removed and cells were grown for 4 additional days. (C) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P p197 RNAi induced and uninduced cells ( n ≥113 for each time point). K, kDNA; N, nucleus. (D) Quantitative analysis of TAC102 in γL262P p197 RNAi cells without tet (no tet), with tet at day five (d5 p.i.) as well as 2 days after removal of tet (post recovery; d2 p.r.) and at day 4 post recovery (d4 p.r.) ( n ≥105 for each time point). (E) Quantitative analysis of the MiRF172–PTP signal in in γL262P p197 RNAi cells as in D ( n ≥105 for each time point). (F) Western blot analysis of γL262P p197 RNAi BSF cells. Total protein isolated from uninduced cells (−tet), cells induced with tet for 5 days (d5 p.i.) and cells released from p197 RNAi at day 2 (d2 p.r.) and day 4 post recovery (d4 p.r.) was used. C-terminally PTP-tagged MiRF172 was detected with the anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. EF1α serves as a loading control. Arrowheads point to the TAC102 and MiRF172 signals. PH, phase contrast. Scale bars: 5 µm.

    Techniques Used: Immunofluorescence, Microscopy, Staining, Western Blot, Isolation

    Quantification of TAC102 in MiRF172 RNAi BSF cells. (A) MiRF172 RNAi BSF cells stained for TAC102 (green) and basal bodies (red) from either uninduced (-tet) or RNAi induced [day (d)3] cells. Pictures show maximum intensity projections from immunofluorescence microscopy image stacks of MiRF172 RNAi BSF T. brucei cells. TAC102 was detected with the anti-TAC102 polyclonal rat antibody and the basal bodies with the monoclonal mouse antibody BBA4. The kDNA and the nucleus were stained with DAPI (cyan). (B) γL262P MiF172 RNAi BSF cells stained for MiRF172–PTP (magenta), TAC102 (green), basal bodies (red) and DAPI (cyan). Proteins and DNA were detected with the same antibodies and reagents as in A. MiRF172–PTP was detected with the anti-Protein A antibody. The pictures show maximum intensity projections as in A. (C) Western blot analysis of γL262P MiRF172 RNAi BSF cells. Total protein isolated from uninduced cells (d0) and cells induced with tet for 3 days (d3). C-terminally PTP-tagged MiRF172 was detected with an anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. Tubulin serves as a loading control. (D) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P MiRF172 RNAi induced and uninduced cells ( n ≥180 for each time point). K, kDNA; N, nucleus. (E) Quantification of TAC102 in γL262P MiRF172 RNAi uninduced (−tet) and cells induced for three days with tet (d3 tet). Black represents the wild-type TAC102 signal and gray stands for a weak TAC102 signal. (F) Quantification of the relative occurrence of the TAC102 signal in γL262P MiRF172 RNAi cells with different kDNA and nucleus DNA content. PH, phase contrast. Scale bars: 5 µm.
    Figure Legend Snippet: Quantification of TAC102 in MiRF172 RNAi BSF cells. (A) MiRF172 RNAi BSF cells stained for TAC102 (green) and basal bodies (red) from either uninduced (-tet) or RNAi induced [day (d)3] cells. Pictures show maximum intensity projections from immunofluorescence microscopy image stacks of MiRF172 RNAi BSF T. brucei cells. TAC102 was detected with the anti-TAC102 polyclonal rat antibody and the basal bodies with the monoclonal mouse antibody BBA4. The kDNA and the nucleus were stained with DAPI (cyan). (B) γL262P MiF172 RNAi BSF cells stained for MiRF172–PTP (magenta), TAC102 (green), basal bodies (red) and DAPI (cyan). Proteins and DNA were detected with the same antibodies and reagents as in A. MiRF172–PTP was detected with the anti-Protein A antibody. The pictures show maximum intensity projections as in A. (C) Western blot analysis of γL262P MiRF172 RNAi BSF cells. Total protein isolated from uninduced cells (d0) and cells induced with tet for 3 days (d3). C-terminally PTP-tagged MiRF172 was detected with an anti-PAP antibody and TAC102 with the anti-TAC102 monoclonal mouse antibody. Tubulin serves as a loading control. (D) Quantification of the relative occurrence of kDNA discs and nuclei in γL262P MiRF172 RNAi induced and uninduced cells ( n ≥180 for each time point). K, kDNA; N, nucleus. (E) Quantification of TAC102 in γL262P MiRF172 RNAi uninduced (−tet) and cells induced for three days with tet (d3 tet). Black represents the wild-type TAC102 signal and gray stands for a weak TAC102 signal. (F) Quantification of the relative occurrence of the TAC102 signal in γL262P MiRF172 RNAi cells with different kDNA and nucleus DNA content. PH, phase contrast. Scale bars: 5 µm.

    Techniques Used: Staining, Immunofluorescence, Microscopy, Western Blot, Isolation

    36) Product Images from "The Role of SDF-1-CXCR4/CXCR7 Axis in the Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stem Cells for Renal Ischemia/Reperfusion Injury"

    Article Title: The Role of SDF-1-CXCR4/CXCR7 Axis in the Therapeutic Effects of Hypoxia-Preconditioned Mesenchymal Stem Cells for Renal Ischemia/Reperfusion Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034608

    Effects of SDF-1-CXCR4/CXCR7 pathway on H 2 O 2 -induced cytotoxicity in MSCs. The standard cytotoxicity tests, including propidium iodide (PI)-based cell viability (A1–A3), MTT assay for mitochondrial viability (B1–B3), LDH assay for membrane damage (C1–C3), were performed. (A1, B1, and C1) MSCs were incubated in H 2 O 2 -conditioned media (250 µM) added with or without SDF-1α (50 ng/ml) for 6 h. The cells incubated in absence of both H 2 O 2 and SDF-1α were used as control. A1 and C1: * P
    Figure Legend Snippet: Effects of SDF-1-CXCR4/CXCR7 pathway on H 2 O 2 -induced cytotoxicity in MSCs. The standard cytotoxicity tests, including propidium iodide (PI)-based cell viability (A1–A3), MTT assay for mitochondrial viability (B1–B3), LDH assay for membrane damage (C1–C3), were performed. (A1, B1, and C1) MSCs were incubated in H 2 O 2 -conditioned media (250 µM) added with or without SDF-1α (50 ng/ml) for 6 h. The cells incubated in absence of both H 2 O 2 and SDF-1α were used as control. A1 and C1: * P

    Techniques Used: MTT Assay, Lactate Dehydrogenase Assay, Incubation

    SDF-1α is upregulated in the kidney of I/R-AKI mice. (A) Representative micrographs of immunohistochemistry for SDF-1α in the kidneys from mice affected by IR-AKI days 1, 2 and 7 after I/R. The kidney sections from mice 24 h after sham surgery were used as control (upper left panel). Original magnification ×200. (B) The kidney cortex lysates from mice affected by sham surgery or I/R-AKI were analyzed by ELISA to determine SDF-1α protein expression at the indicated periods of post-surgery time. * P
    Figure Legend Snippet: SDF-1α is upregulated in the kidney of I/R-AKI mice. (A) Representative micrographs of immunohistochemistry for SDF-1α in the kidneys from mice affected by IR-AKI days 1, 2 and 7 after I/R. The kidney sections from mice 24 h after sham surgery were used as control (upper left panel). Original magnification ×200. (B) The kidney cortex lysates from mice affected by sham surgery or I/R-AKI were analyzed by ELISA to determine SDF-1α protein expression at the indicated periods of post-surgery time. * P

    Techniques Used: Mouse Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing

    A model of regenerative potential of HP-MSCs in repair of I/R-AKI. Chemokine SDF-1 expression is upregulated in postischemic kidneys. HP enhances the expression of both SDF-1 receptors, CXCR4 and CXCR7, in MSCs. Intravenously injected HP-MSCs are recruited to the ischemic kidney and localized within the injured capillaries and in the interstitium through SDF-1α-CXCR4 interaction. The binding of SDF-1 to both CXCR4 and CXCR7 is responsible for the production of paracrine mediators, including VEGF, β-FGF, IGF-1 and HGF that exert mitogenic, anti-apoptotic, pro-angiogenic, and anti-inflammatory effects.
    Figure Legend Snippet: A model of regenerative potential of HP-MSCs in repair of I/R-AKI. Chemokine SDF-1 expression is upregulated in postischemic kidneys. HP enhances the expression of both SDF-1 receptors, CXCR4 and CXCR7, in MSCs. Intravenously injected HP-MSCs are recruited to the ischemic kidney and localized within the injured capillaries and in the interstitium through SDF-1α-CXCR4 interaction. The binding of SDF-1 to both CXCR4 and CXCR7 is responsible for the production of paracrine mediators, including VEGF, β-FGF, IGF-1 and HGF that exert mitogenic, anti-apoptotic, pro-angiogenic, and anti-inflammatory effects.

    Techniques Used: Expressing, Injection, Binding Assay

    Effects of SDF-1-CXCR4/CXCR7 pathway on MSC chemotaxis in vitro. (A) The chemotaxis in response to SDF-1α (10 ng/ml for 12 h) was performed in the NP-MSCs and HP-MSCs treated with a neutralizing anti-CXCR4 antibody, an anti-CXCR7 antibody, and the respective isotype-matched control antibodies. * P
    Figure Legend Snippet: Effects of SDF-1-CXCR4/CXCR7 pathway on MSC chemotaxis in vitro. (A) The chemotaxis in response to SDF-1α (10 ng/ml for 12 h) was performed in the NP-MSCs and HP-MSCs treated with a neutralizing anti-CXCR4 antibody, an anti-CXCR7 antibody, and the respective isotype-matched control antibodies. * P

    Techniques Used: Chemotaxis Assay, In Vitro

    Effects of SDF-1-CXCR4/CXCR7 pathway on MSC paracrine actions. ELISA was performed to determine production of VEGF, β-FGF, IGF-1 and HGF from MSCs stimulated by hypoxia (3% O 2 ) or/and SDF-1α (50 ng/ml). The cells stimulated by neither hypoxia nor SDF-1α were used as control. (A) MSCs were stimulated with hypoxia or/and SDF-1α. * P
    Figure Legend Snippet: Effects of SDF-1-CXCR4/CXCR7 pathway on MSC paracrine actions. ELISA was performed to determine production of VEGF, β-FGF, IGF-1 and HGF from MSCs stimulated by hypoxia (3% O 2 ) or/and SDF-1α (50 ng/ml). The cells stimulated by neither hypoxia nor SDF-1α were used as control. (A) MSCs were stimulated with hypoxia or/and SDF-1α. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effects of HP on the expression of SDF-1α, CXCR4, CXCR7 in MSCs. (A) Semiquantitative RT-PCR was used for the analysis of SDF-1α, CXCR4 and CXCR7 mRNA levels in MSCs. GAPDH was used as a control. Lane 1 indicates bone marrow mononuclear cells (BMMCs); lanes 2 to 4, MSC cultures at passage 1 to 3; and lane 5, MSCs at passage 3 and exposed to hypoxia (3% O 2 ) for 24 h. (B) Western blot analysis was performed to detect CXCR4, CXCR7 and SDF-1α protein expression. β-actin was used as a control. Lanes 1 indicates BMMCs; lanes 2 to 5, MSC cultures at passage 1 to 4; and lane 6, MSCs at the third passage to hypoxia for 24 h. (C) FCM was used to detect extracellular expression of CXCR4 or CXCR7 in MSCs exposed to the indicated periods of hypoxia. * P
    Figure Legend Snippet: Effects of HP on the expression of SDF-1α, CXCR4, CXCR7 in MSCs. (A) Semiquantitative RT-PCR was used for the analysis of SDF-1α, CXCR4 and CXCR7 mRNA levels in MSCs. GAPDH was used as a control. Lane 1 indicates bone marrow mononuclear cells (BMMCs); lanes 2 to 4, MSC cultures at passage 1 to 3; and lane 5, MSCs at passage 3 and exposed to hypoxia (3% O 2 ) for 24 h. (B) Western blot analysis was performed to detect CXCR4, CXCR7 and SDF-1α protein expression. β-actin was used as a control. Lanes 1 indicates BMMCs; lanes 2 to 5, MSC cultures at passage 1 to 4; and lane 6, MSCs at the third passage to hypoxia for 24 h. (C) FCM was used to detect extracellular expression of CXCR4 or CXCR7 in MSCs exposed to the indicated periods of hypoxia. * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    The role of SDF-1-CXCR4/CXCR7 pathway on the homing of HP-MSCs toward ischemic kidneys. (A) ELISA analysis was performed to determine production of SDF-1α from primary TECs exposed to hypoxia/reoxygenation in vitro. The cells without hypoxia/reoxygenation stimulation were used as control. * P
    Figure Legend Snippet: The role of SDF-1-CXCR4/CXCR7 pathway on the homing of HP-MSCs toward ischemic kidneys. (A) ELISA analysis was performed to determine production of SDF-1α from primary TECs exposed to hypoxia/reoxygenation in vitro. The cells without hypoxia/reoxygenation stimulation were used as control. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, In Vitro

    37) Product Images from "Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells"

    Article Title: Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-42-111

    Analysis of toll-like receptor 3 (TLR3) expression in porcine intestinal epithelial (PIE) cells . (A) Flow cytometric analysis of TLR3 in PIE cells. Histograms show flow cytometric analysis for TLR3 staining as follows: intracellular (open histogram), cell surface (broken lines) and isotype-matched controls (shaded histograms). The analysis of intracellular and cell surface expression of TLR3 is presented as the log of mean fluorescence intensity (MFI). The results represent three independent experiments. (B) Confocal microscopic analysis of the subcellular localization of TLR3 in PIE cells. Fixed and permeabilized cells were stained with anti-mouse TLR3(unlabeled) rabbit-IgG and goat anti-rabbit IgG (H+L) Alexa Fluor488 and Early Endosomes-RFP BacMam. The merged color (yellow) is indicative of co-localization. Bar, 2 um. PIE cells stained with rabbit IgG-Alexa Fluor488 Isotype were used as controls.
    Figure Legend Snippet: Analysis of toll-like receptor 3 (TLR3) expression in porcine intestinal epithelial (PIE) cells . (A) Flow cytometric analysis of TLR3 in PIE cells. Histograms show flow cytometric analysis for TLR3 staining as follows: intracellular (open histogram), cell surface (broken lines) and isotype-matched controls (shaded histograms). The analysis of intracellular and cell surface expression of TLR3 is presented as the log of mean fluorescence intensity (MFI). The results represent three independent experiments. (B) Confocal microscopic analysis of the subcellular localization of TLR3 in PIE cells. Fixed and permeabilized cells were stained with anti-mouse TLR3(unlabeled) rabbit-IgG and goat anti-rabbit IgG (H+L) Alexa Fluor488 and Early Endosomes-RFP BacMam. The merged color (yellow) is indicative of co-localization. Bar, 2 um. PIE cells stained with rabbit IgG-Alexa Fluor488 Isotype were used as controls.

    Techniques Used: Expressing, Flow Cytometry, Staining, Fluorescence

    38) Product Images from "Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival"

    Article Title: Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival

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

    doi: 10.1073/pnas.1117374108

    Titration of CD45 expression differentially regulates AgR signaling in T and B cells. ( A ) CD19 + , CD4 + , and CD8 + LN cells from an allelic series of mice generated by crossing CD45 WT, lightning (L), and null alleles, and CD45 ‘H’ Tg were
    Figure Legend Snippet: Titration of CD45 expression differentially regulates AgR signaling in T and B cells. ( A ) CD19 + , CD4 + , and CD8 + LN cells from an allelic series of mice generated by crossing CD45 WT, lightning (L), and null alleles, and CD45 ‘H’ Tg were

    Techniques Used: Titration, Expressing, Mouse Assay, Generated

    High CD45 expression negatively regulates follicular B-cell development. ( A ) Graph showing total splenic cells from allelic series mice. Values are mean ± SEM of between four and six biological replicates. ( B ) Graph presenting total splenic CD19+
    Figure Legend Snippet: High CD45 expression negatively regulates follicular B-cell development. ( A ) Graph showing total splenic cells from allelic series mice. Values are mean ± SEM of between four and six biological replicates. ( B ) Graph presenting total splenic CD19+

    Techniques Used: Expressing, Mouse Assay

    39) Product Images from "Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival"

    Article Title: Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival

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

    doi: 10.1073/pnas.1117374108

    Titration of CD45 expression differentially regulates AgR signaling in T and B cells. ( A ) CD19 + , CD4 + , and CD8 + LN cells from an allelic series of mice generated by crossing CD45 WT, lightning (L), and null alleles, and CD45 ‘H’ Tg were
    Figure Legend Snippet: Titration of CD45 expression differentially regulates AgR signaling in T and B cells. ( A ) CD19 + , CD4 + , and CD8 + LN cells from an allelic series of mice generated by crossing CD45 WT, lightning (L), and null alleles, and CD45 ‘H’ Tg were

    Techniques Used: Titration, Expressing, Mouse Assay, Generated

    40) Product Images from "Gene expression profiling for nitric oxide prodrug JS-K to kill HL-60 myeloid leukemia cells"

    Article Title: Gene expression profiling for nitric oxide prodrug JS-K to kill HL-60 myeloid leukemia cells

    Journal: Genomics

    doi: 10.1016/j.ygeno.2009.03.005

    Representative confocal image analysis of the expression of caspase-3, CD14, c-myc, and TIMP1. HL-60 cells were treated with 1.0 μM JS-K or leave untreated for 24 hr. Cells were then harvested, fixed, permeabilized, and incubated with specific
    Figure Legend Snippet: Representative confocal image analysis of the expression of caspase-3, CD14, c-myc, and TIMP1. HL-60 cells were treated with 1.0 μM JS-K or leave untreated for 24 hr. Cells were then harvested, fixed, permeabilized, and incubated with specific

    Techniques Used: Expressing, Incubation

    Related Articles

    Staining:

    Article Title: Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells
    Article Snippet: .. Cells stained with irrelevant mouse IgG2b-FITC (11-4732, eBioscience), IgG1-PerCP/Cy5.5 (45-4714, eBioscience), IgG2b-PE (12-4732, eBioscience), IgG2a-PE (12-4724, eBioscience), IgG1-PE (12-4714, eBioscience) and rabbit IgG-Alexa Fluor488 isotype control (4340S, Cell Signaling Technology Japan KK, Tokyo, Japan) antibodies were included as isotype controls. ..

    Article Title: Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells
    Article Snippet: .. Cells stained with irrelevant mouse IgG-FITC, IgG2b-FITC, IgG2a-PerCP, IgG2b-PE, IgG2a-PE, or IgG1-PE antibodies (eBioscience, San Diego, CA) were included as isotype controls. .. Analysis of the stained cells was performed using a FACSCalibur flow cytometer (BD, Franklin Lakes, NJ), which was equipped with Cell-Quest software.

    Incubation:

    Article Title: Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency
    Article Snippet: .. For characterization of the splenic B cell compartment, 5-µm sections of the spleens from 8-wk-old mut/mut and +/+ mice (n = 4 for each) were incubated with B220–Alexa Fluor 647, IgG–Alexa Fluor 488, IgG2a-PE (Invitrogen), and peanut agglutinin–FITC (Vector Laboratories). .. To detect immune complexes in the kidney glomeruli, 5-µm frozen sections were prepared from the kidneys of four 12-wk-old mut/mut , two 12-wk-old +/+, three 8-mo-old mut/mut , and two 8-mo-old +/+ mice.

    other:

    Article Title: Varicella-Zoster Virus Infection of Human Dendritic Cells and Transmission to T Cells: Implications for Virus Dissemination in the Host
    Article Snippet: Monoclonal antibodies specific for human MHC class II DR (clone TU36; R-phycoerythrin [PE] conjugated), human CD14 (clone TUK4; fluorescein isothiocyanate [FITC] conjugated), human CD40 (clone 14G7; unconjugated), human CD71 (clone T56/14), human CD3 (clone S4.1; tricolor [TC] conjugated), human CD4 (clone S3.5; PE conjugated), human CD8 (clone 3B5; TC conjugated), PE-conjugated goat anti-human immunoglobulin G (IgG), FITC-conjugated goat anti-human IgG, TC-conjugated goat anti-human IgG, mouse IgG, mouse IgG2a-PE, mouse IgG2a-TC, and mouse IgG2a-FITC were obtained from Caltag Laboratories (San Francisco, Calif.).

    Mouse Assay:

    Article Title: Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency
    Article Snippet: .. For characterization of the splenic B cell compartment, 5-µm sections of the spleens from 8-wk-old mut/mut and +/+ mice (n = 4 for each) were incubated with B220–Alexa Fluor 647, IgG–Alexa Fluor 488, IgG2a-PE (Invitrogen), and peanut agglutinin–FITC (Vector Laboratories). .. To detect immune complexes in the kidney glomeruli, 5-µm frozen sections were prepared from the kidneys of four 12-wk-old mut/mut , two 12-wk-old +/+, three 8-mo-old mut/mut , and two 8-mo-old +/+ mice.

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  • 99
    Thermo Fisher biotinylated goat anti rabbit igg
    Comparison of the binding of C-HBV to human (hPBMCs) with sheep (sPBMCS) peripheral blood mononuclear cells. PBMCs were isolated from either sheep or human blood and incubated for 1 h at 4°C with C-HBV (1–50 μg/mL). Following labeling with <t>biotinylated</t> anti-mouse <t>IgG</t> mAb and SA-PE-Cy5, bound protein was quantified by flow cytometry and expressed as the relative mean fluorescence intensity (MFI) of labeled cells.
    Biotinylated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated goat anti rabbit igg/product/Thermo Fisher
    Average 99 stars, based on 99 article reviews
    Price from $9.99 to $1999.99
    biotinylated goat anti rabbit igg - by Bioz Stars, 2020-09
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    84
    Thermo Fisher final concentration alexa fluor 488 conjugated goat anti rabbit igg
    PG biogenesis and Z-ring placement are largely unaffected in the Δ zicK Δ zacK mutant ( A ) <t>Alexa</t> Fluor-488 and bright field micrographs of Anabaena WT and Δ zicK Δ zacK mutant subjected to anti-FtsZ immunofluorescence. ( B ) Merged BODIPY™ FL Vancomycin (Van-FL) fluorescence and chlorophyll autofluorescence micrographs of Anabaena WT and the Δ zicK Δ zacK mutant stained with Van-FL. As a result of the low Van-FL staining and for better visibility, Van-FL fluorescence signal in Δ zicK Δ zacK mutant was artificially increased about twofold after image acquisition (note: this increase was not used for the fluorescence intensity measurement in ( C )). Scale bars: ( A , B ) 5 μm. ( C ) Arithmetic mean fluorescence intensities of n=200 cell septa from ( B ). Values indicated with * are significantly different from the WT. **: P
    Final Concentration Alexa Fluor 488 Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/final concentration alexa fluor 488 conjugated goat anti rabbit igg/product/Thermo Fisher
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    final concentration alexa fluor 488 conjugated goat anti rabbit igg - by Bioz Stars, 2020-09
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    94
    Thermo Fisher goat anti rabbit igg
    TGEV infection induced EGFR internalization. (A) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. Then stained for fluorescence microscope using rabbit anti-EGFR pAb followed by <t>DyLight</t> 594-conjugated goat anti-rabbit <t>IgG,</t> Mock-infected cells served as controls. EGFR distribution was observed by confocal microscope. (B) Three-dimensional rendering of representative images obtained using Imaris 7.2 software. (C) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (D) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD, from three independent experiments. (scale bar = 20 µm).
    Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of the binding of C-HBV to human (hPBMCs) with sheep (sPBMCS) peripheral blood mononuclear cells. PBMCs were isolated from either sheep or human blood and incubated for 1 h at 4°C with C-HBV (1–50 μg/mL). Following labeling with biotinylated anti-mouse IgG mAb and SA-PE-Cy5, bound protein was quantified by flow cytometry and expressed as the relative mean fluorescence intensity (MFI) of labeled cells.

    Journal: Human Vaccines & Immunotherapeutics

    Article Title: A dendritic cell-targeted chimeric hepatitis B virus immunotherapeutic vaccine induces both cellular and humoral immune responses in vivo

    doi: 10.1080/21645515.2019.1689081

    Figure Lengend Snippet: Comparison of the binding of C-HBV to human (hPBMCs) with sheep (sPBMCS) peripheral blood mononuclear cells. PBMCs were isolated from either sheep or human blood and incubated for 1 h at 4°C with C-HBV (1–50 μg/mL). Following labeling with biotinylated anti-mouse IgG mAb and SA-PE-Cy5, bound protein was quantified by flow cytometry and expressed as the relative mean fluorescence intensity (MFI) of labeled cells.

    Article Snippet: Following overnight incubation at 37°C, cells were lysed with distilled water and captured IFN-γ visualized with rabbit anti-bovine IFN-γ antisera (Rockland Immunochemicals, 201-401-C41) and biotinylated-goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, 31820).

    Techniques: Binding Assay, Isolation, Incubation, Labeling, Flow Cytometry, Fluorescence

    PG biogenesis and Z-ring placement are largely unaffected in the Δ zicK Δ zacK mutant ( A ) Alexa Fluor-488 and bright field micrographs of Anabaena WT and Δ zicK Δ zacK mutant subjected to anti-FtsZ immunofluorescence. ( B ) Merged BODIPY™ FL Vancomycin (Van-FL) fluorescence and chlorophyll autofluorescence micrographs of Anabaena WT and the Δ zicK Δ zacK mutant stained with Van-FL. As a result of the low Van-FL staining and for better visibility, Van-FL fluorescence signal in Δ zicK Δ zacK mutant was artificially increased about twofold after image acquisition (note: this increase was not used for the fluorescence intensity measurement in ( C )). Scale bars: ( A , B ) 5 μm. ( C ) Arithmetic mean fluorescence intensities of n=200 cell septa from ( B ). Values indicated with * are significantly different from the WT. **: P

    Journal: bioRxiv

    Article Title: Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacterium Anabaena sp. PCC 7120

    doi: 10.1101/553073

    Figure Lengend Snippet: PG biogenesis and Z-ring placement are largely unaffected in the Δ zicK Δ zacK mutant ( A ) Alexa Fluor-488 and bright field micrographs of Anabaena WT and Δ zicK Δ zacK mutant subjected to anti-FtsZ immunofluorescence. ( B ) Merged BODIPY™ FL Vancomycin (Van-FL) fluorescence and chlorophyll autofluorescence micrographs of Anabaena WT and the Δ zicK Δ zacK mutant stained with Van-FL. As a result of the low Van-FL staining and for better visibility, Van-FL fluorescence signal in Δ zicK Δ zacK mutant was artificially increased about twofold after image acquisition (note: this increase was not used for the fluorescence intensity measurement in ( C )). Scale bars: ( A , B ) 5 μm. ( C ) Arithmetic mean fluorescence intensities of n=200 cell septa from ( B ). Values indicated with * are significantly different from the WT. **: P

    Article Snippet: 7.5 μg ml−1 (final concentration) Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fischer Scientific) in blocking buffer was added to the cells and incubated for 1 h at RT in the dark in a self-made humidity chamber.

    Techniques: Mutagenesis, Immunofluorescence, Fluorescence, Staining

    TGEV infection induced EGFR internalization. (A) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. Then stained for fluorescence microscope using rabbit anti-EGFR pAb followed by DyLight 594-conjugated goat anti-rabbit IgG, Mock-infected cells served as controls. EGFR distribution was observed by confocal microscope. (B) Three-dimensional rendering of representative images obtained using Imaris 7.2 software. (C) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (D) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD, from three independent experiments. (scale bar = 20 µm).

    Journal: Virology

    Article Title: Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry

    doi: 10.1016/j.virol.2018.05.009

    Figure Lengend Snippet: TGEV infection induced EGFR internalization. (A) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. Then stained for fluorescence microscope using rabbit anti-EGFR pAb followed by DyLight 594-conjugated goat anti-rabbit IgG, Mock-infected cells served as controls. EGFR distribution was observed by confocal microscope. (B) Three-dimensional rendering of representative images obtained using Imaris 7.2 software. (C) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (D) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD, from three independent experiments. (scale bar = 20 µm).

    Article Snippet: Goat anti-rabbit IgG (H + L) secondary Antibody, DyLight 594 conjugate, goat anti-mouse IgG (H + L) secondary Antibody, DyLight 488 conjugate (ThermoFisher Scientific).

    Techniques: Infection, Cell Culture, Staining, Fluorescence, Microscopy, Software

    TGEV infection causes the co-localization of APN and EGFR. IPEC-J2 cells were infected with TGEV (MOI = 2) and cultured for 30 min, then stained for fluorescence microscopy using mouse anti-APN pAb, followed by DyLight 488-conjugated goat anti-mouse IgG and rabbit anti-p-EGFR mAb, followed by DyLight 594-conjugated goat anti-rabbit IgG. Mock-infected cells served as controls. The data shown are from two independent experiments.

    Journal: Virology

    Article Title: Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry

    doi: 10.1016/j.virol.2018.05.009

    Figure Lengend Snippet: TGEV infection causes the co-localization of APN and EGFR. IPEC-J2 cells were infected with TGEV (MOI = 2) and cultured for 30 min, then stained for fluorescence microscopy using mouse anti-APN pAb, followed by DyLight 488-conjugated goat anti-mouse IgG and rabbit anti-p-EGFR mAb, followed by DyLight 594-conjugated goat anti-rabbit IgG. Mock-infected cells served as controls. The data shown are from two independent experiments.

    Article Snippet: Goat anti-rabbit IgG (H + L) secondary Antibody, DyLight 594 conjugate, goat anti-mouse IgG (H + L) secondary Antibody, DyLight 488 conjugate (ThermoFisher Scientific).

    Techniques: Infection, Cell Culture, Staining, Fluorescence, Microscopy