gst nlg1 wt  (Millipore)


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    Structured Review

    Millipore gst nlg1 wt
    Identification of tyrosine kinases that phosphorylate <t>Nlg1.</t> a , b In vitro kinase assay. Purified tyrosine kinases <t>(GST-FGFR1,</t> -TrkB, or -TrkC) were incubated in the presence of ATP with GST-Nlg1, GST-Nlg1-Y782A, or a positive substrate (NTRK), and run on a polyacrylamide gel. a pTyr immunoblot. Note the phosphorylation of NTRK (blue arrow) and GST-Nlg1 (red arrow) but not GST-Nlg1-Y782A by the three kinases. Note the autophosphorylation of the three kinases (green arrow). b Corresponding Coomassie gel. GST-Nlg1 shows several bands, corresponding to partial degradation. The strongest band at 40 kD contains the gephyrin-binding motif. The NTRK and kinases are present in low amounts and barely appear on the gel. c Protein extracts from COS cells expressing Nlg1-WT, Nlg1-Y782A, with or without FGFR1, TrkB, or TrkC, were immunoprecipitated with Nlg1 antibodies. Some cells were pretreated with FGFR1 or pan-Trk inhibitors. pTyr and Nlg1 immunoblots are shown. d pTyr signals normalized to Nlg1 levels in the different conditions (number of experiments within bars). e , f Protein extracts from cortical cultures at DIV 10–14 pretreated with the kinase inhibitors for 24 h were immunoprecipitated with Nlg1, Nlg2, or Nlg3 antibodies. pTyr and Nlg1/2/3 immunoblots are shown, respectively. g Average pTyr signals for the different inhibitors, normalized to the control, untreated condition (number of experiments within bars). h CA1 neurons from Nlg1 KO slices were electroporated with Nlg1-WT or Nlg1-Y782A, and AMPAR-mediated EPSCs were recorded upon stimulation of Schaffer’s collaterals, in comparison to unelectroporated neighboring neurons (black traces). Organotypic cultures were treated or not with 1 µM GNF5837 for 7 days before the recordings. i Average AMPAR-mediated EPSCs amplitude in the 3 conditions, normalized to non-electroporated controls (number of pairs indicated within the bars, from 3 independent experiments). Data in graphs d and g were compared by one-way ANOVA followed by Bonferroni post hoc test (ns: not significant, * P
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    Images

    1) Product Images from "A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation"

    Article Title: A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06220-2

    Identification of tyrosine kinases that phosphorylate Nlg1. a , b In vitro kinase assay. Purified tyrosine kinases (GST-FGFR1, -TrkB, or -TrkC) were incubated in the presence of ATP with GST-Nlg1, GST-Nlg1-Y782A, or a positive substrate (NTRK), and run on a polyacrylamide gel. a pTyr immunoblot. Note the phosphorylation of NTRK (blue arrow) and GST-Nlg1 (red arrow) but not GST-Nlg1-Y782A by the three kinases. Note the autophosphorylation of the three kinases (green arrow). b Corresponding Coomassie gel. GST-Nlg1 shows several bands, corresponding to partial degradation. The strongest band at 40 kD contains the gephyrin-binding motif. The NTRK and kinases are present in low amounts and barely appear on the gel. c Protein extracts from COS cells expressing Nlg1-WT, Nlg1-Y782A, with or without FGFR1, TrkB, or TrkC, were immunoprecipitated with Nlg1 antibodies. Some cells were pretreated with FGFR1 or pan-Trk inhibitors. pTyr and Nlg1 immunoblots are shown. d pTyr signals normalized to Nlg1 levels in the different conditions (number of experiments within bars). e , f Protein extracts from cortical cultures at DIV 10–14 pretreated with the kinase inhibitors for 24 h were immunoprecipitated with Nlg1, Nlg2, or Nlg3 antibodies. pTyr and Nlg1/2/3 immunoblots are shown, respectively. g Average pTyr signals for the different inhibitors, normalized to the control, untreated condition (number of experiments within bars). h CA1 neurons from Nlg1 KO slices were electroporated with Nlg1-WT or Nlg1-Y782A, and AMPAR-mediated EPSCs were recorded upon stimulation of Schaffer’s collaterals, in comparison to unelectroporated neighboring neurons (black traces). Organotypic cultures were treated or not with 1 µM GNF5837 for 7 days before the recordings. i Average AMPAR-mediated EPSCs amplitude in the 3 conditions, normalized to non-electroporated controls (number of pairs indicated within the bars, from 3 independent experiments). Data in graphs d and g were compared by one-way ANOVA followed by Bonferroni post hoc test (ns: not significant, * P
    Figure Legend Snippet: Identification of tyrosine kinases that phosphorylate Nlg1. a , b In vitro kinase assay. Purified tyrosine kinases (GST-FGFR1, -TrkB, or -TrkC) were incubated in the presence of ATP with GST-Nlg1, GST-Nlg1-Y782A, or a positive substrate (NTRK), and run on a polyacrylamide gel. a pTyr immunoblot. Note the phosphorylation of NTRK (blue arrow) and GST-Nlg1 (red arrow) but not GST-Nlg1-Y782A by the three kinases. Note the autophosphorylation of the three kinases (green arrow). b Corresponding Coomassie gel. GST-Nlg1 shows several bands, corresponding to partial degradation. The strongest band at 40 kD contains the gephyrin-binding motif. The NTRK and kinases are present in low amounts and barely appear on the gel. c Protein extracts from COS cells expressing Nlg1-WT, Nlg1-Y782A, with or without FGFR1, TrkB, or TrkC, were immunoprecipitated with Nlg1 antibodies. Some cells were pretreated with FGFR1 or pan-Trk inhibitors. pTyr and Nlg1 immunoblots are shown. d pTyr signals normalized to Nlg1 levels in the different conditions (number of experiments within bars). e , f Protein extracts from cortical cultures at DIV 10–14 pretreated with the kinase inhibitors for 24 h were immunoprecipitated with Nlg1, Nlg2, or Nlg3 antibodies. pTyr and Nlg1/2/3 immunoblots are shown, respectively. g Average pTyr signals for the different inhibitors, normalized to the control, untreated condition (number of experiments within bars). h CA1 neurons from Nlg1 KO slices were electroporated with Nlg1-WT or Nlg1-Y782A, and AMPAR-mediated EPSCs were recorded upon stimulation of Schaffer’s collaterals, in comparison to unelectroporated neighboring neurons (black traces). Organotypic cultures were treated or not with 1 µM GNF5837 for 7 days before the recordings. i Average AMPAR-mediated EPSCs amplitude in the 3 conditions, normalized to non-electroporated controls (number of pairs indicated within the bars, from 3 independent experiments). Data in graphs d and g were compared by one-way ANOVA followed by Bonferroni post hoc test (ns: not significant, * P

    Techniques Used: In Vitro, Kinase Assay, Purification, Incubation, Binding Assay, Expressing, Immunoprecipitation, Western Blot

    Nlg1 mutants differentially associate with gephyrin and PSD-95 scaffolds. a Schematics of Nlg1 structure. The sequence of the gephyrin-binding motif (blue) is shown with Y782 in red; AchE: acetylcholine esterase-like domain; TM: transmembrane domain. b Coomassie gel loaded with GST or GST-Nlg1 fusion proteins (Nlg1-WT, Y782A, or Y782F). These proteins migrate as three major bands between 25 kD and 50 kD, suggesting that they experience some degradation. The upper bands at 50 kD corresponding to the full length molecule are expected to bind PSD-95. This upper band plus the major band at ~40 kD are expected to bind gephyrin. c , d Immunoblots of gephyrin-Venus and PSD-95-mCherry pulled down by GST-Nlg1 proteins, respectively, and corresponding graphs of bound gephyrin and PSD-95, normalized to GST-Nlg1-WT (mean ± SEM of 2 independent experiments). e , g Dendrites from DIV 14–15 cultured neurons electroporated at DIV 0 with AP-tagged Nlg1-WT, -Y782A, or -Y782F (red) along with BirA ER and GFP (blue). Surface Nlg1 was labeled with Atto647-conjugated mSA (red) and endogenous gephyrin or PSD-95 were immunostained, respectively (green). Scale bars, 10 µm. f , h Percentage of surface Nlg1 clusters positive for gephyrin or PSD-95 for the three conditions (number of cells indicated within the bars, from 3 independent experiments). i Representative trajectories of individual Nlg1 molecules tracked by uPAINT (see Methods) at the surface of neurons at DIV 14–15 expressing Nlg1-WT, -Y782A, or -Y782F along with BirA ER and Homer1c-GFP (white), sparsely labeled with Atto594-conjugated mSA. Magenta and green represent fast ( D > 0.01 µm²/s) and slow ( D ≤ 0.01 µm²/s) diffusing molecules, respectively. Scale bar, 2 µm. j Distributions of individual diffusion coefficients in log scale (26013, 28756, and 17894 trajectories from 7, 10, and 9 cells for Nlg1-WT, Nlg1-Y782A, and Nlg1-Y782F, respectively). k Median diffusion coefficient for the 3 conditions (from one experiment). Data in graphs f , h , and k were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (* P
    Figure Legend Snippet: Nlg1 mutants differentially associate with gephyrin and PSD-95 scaffolds. a Schematics of Nlg1 structure. The sequence of the gephyrin-binding motif (blue) is shown with Y782 in red; AchE: acetylcholine esterase-like domain; TM: transmembrane domain. b Coomassie gel loaded with GST or GST-Nlg1 fusion proteins (Nlg1-WT, Y782A, or Y782F). These proteins migrate as three major bands between 25 kD and 50 kD, suggesting that they experience some degradation. The upper bands at 50 kD corresponding to the full length molecule are expected to bind PSD-95. This upper band plus the major band at ~40 kD are expected to bind gephyrin. c , d Immunoblots of gephyrin-Venus and PSD-95-mCherry pulled down by GST-Nlg1 proteins, respectively, and corresponding graphs of bound gephyrin and PSD-95, normalized to GST-Nlg1-WT (mean ± SEM of 2 independent experiments). e , g Dendrites from DIV 14–15 cultured neurons electroporated at DIV 0 with AP-tagged Nlg1-WT, -Y782A, or -Y782F (red) along with BirA ER and GFP (blue). Surface Nlg1 was labeled with Atto647-conjugated mSA (red) and endogenous gephyrin or PSD-95 were immunostained, respectively (green). Scale bars, 10 µm. f , h Percentage of surface Nlg1 clusters positive for gephyrin or PSD-95 for the three conditions (number of cells indicated within the bars, from 3 independent experiments). i Representative trajectories of individual Nlg1 molecules tracked by uPAINT (see Methods) at the surface of neurons at DIV 14–15 expressing Nlg1-WT, -Y782A, or -Y782F along with BirA ER and Homer1c-GFP (white), sparsely labeled with Atto594-conjugated mSA. Magenta and green represent fast ( D > 0.01 µm²/s) and slow ( D ≤ 0.01 µm²/s) diffusing molecules, respectively. Scale bar, 2 µm. j Distributions of individual diffusion coefficients in log scale (26013, 28756, and 17894 trajectories from 7, 10, and 9 cells for Nlg1-WT, Nlg1-Y782A, and Nlg1-Y782F, respectively). k Median diffusion coefficient for the 3 conditions (from one experiment). Data in graphs f , h , and k were compared by a Kruskal–Wallis test followed by Dunn’s multiple comparison test (* P

    Techniques Used: Sequencing, Binding Assay, Western Blot, Cell Culture, Labeling, Expressing, Diffusion-based Assay

    2) Product Images from "Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells"

    Article Title: Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205108

    Expression of markers of interest during differentiation of mouse ESCs Ccbe1 in cardiac progenitors isolated from differentiating mouse ESCs and embryos at E9.5. (A-F) Samples were collected from undifferentiated cells (Und) and at days 2, 4, 6, 8 and 10 of differentiating RG mouse ESCs. Expression is presented as fold change relative to undifferentiated cells. Data presented as the mean + SEM of two biological replicates in technical qPCR triplicates. (G-M) Ccbe1 expression was analyzed in isolated populations, defined as FHF G+R- population, SHF G+R+ and G-R+ populations and control G-R- population at (G) day 6, (H) day 8 and (I) day 11 from differentiating mouse ESCs, and from (K) E9.5 mouse embryos. Expression is represented as fold change relative to the control G-R-population. (J) Analysis of Ccbe1 expression in the control G-R- population at day 6, 8 and 11 relative to the expression at day 6. The identity of the sorted populations at day 6 was confirmed by analyzing the expression of (L) Ist1and (M) Nkx2.5. Mean + SEM of two biological replicates.
    Figure Legend Snippet: Expression of markers of interest during differentiation of mouse ESCs Ccbe1 in cardiac progenitors isolated from differentiating mouse ESCs and embryos at E9.5. (A-F) Samples were collected from undifferentiated cells (Und) and at days 2, 4, 6, 8 and 10 of differentiating RG mouse ESCs. Expression is presented as fold change relative to undifferentiated cells. Data presented as the mean + SEM of two biological replicates in technical qPCR triplicates. (G-M) Ccbe1 expression was analyzed in isolated populations, defined as FHF G+R- population, SHF G+R+ and G-R+ populations and control G-R- population at (G) day 6, (H) day 8 and (I) day 11 from differentiating mouse ESCs, and from (K) E9.5 mouse embryos. Expression is represented as fold change relative to the control G-R-population. (J) Analysis of Ccbe1 expression in the control G-R- population at day 6, 8 and 11 relative to the expression at day 6. The identity of the sorted populations at day 6 was confirmed by analyzing the expression of (L) Ist1and (M) Nkx2.5. Mean + SEM of two biological replicates.

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

    3) Product Images from "Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2"

    Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.05.018

    SETD1A and FANCD2 Promote H3 Mobility to Protect Fork Integrity (A) U-2-OS cells were transfected with the indicated siRNAs and treated as in Figure 3 . Arrows indicate mean ratios. (B) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were transfected with the indicated siRNAs and treated as above. (C) U-2-OS cells from (A) were transfected with SNAP-tagged H3.1 and analyzed to reveal levels of pre-existing SNAP-H3.1 (Pulse), background fluorescence (Quench-Pulse), and new H3.1 after a 2-hr release into HU (Quench-HU-Pulse). Cells were analyzed by fluorescence microscopy, and fluorescence intensity per nucleus was quantified using ImageJ. (D) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were left untreated or exposed to MMC for 24 hr and analyzed by FRAP. (E) WT DT40 cells or FANCD2 −/− cells expressing the indicated GFP-tagged chFANCD2 variants were treated as described in Figure 3 . Arrows indicate mean ratios. (F) Stable U-2-OS-GFP-RAD51 cells were co-transfected with the indicated siRNA and plasmids encoding either WT or chaperone-dead (R302W) mCherry-FANCD2 and exposed to MMC, and the mobility of GFP-RAD51 was analyzed by FRAP. (G) Model for the role of SET1A-dependent H3K4 methylation in protecting stalled replication forks. Upon fork reversal during replication stress, H3K4me1 by SETD1A acts to restrict CHD4 localization and promote FANCD2-dependent histone mobility. This chaperone activity is vital to stabilize RAD51 nucleofilaments, preventing nucleolytic degradation of stalled forks by DNA2. The plots in (D) and (F) represent mean ± SD relative fluorescence intensities from n = 21–26 and 25–48 cells, respectively. ∗ p
    Figure Legend Snippet: SETD1A and FANCD2 Promote H3 Mobility to Protect Fork Integrity (A) U-2-OS cells were transfected with the indicated siRNAs and treated as in Figure 3 . Arrows indicate mean ratios. (B) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were transfected with the indicated siRNAs and treated as above. (C) U-2-OS cells from (A) were transfected with SNAP-tagged H3.1 and analyzed to reveal levels of pre-existing SNAP-H3.1 (Pulse), background fluorescence (Quench-Pulse), and new H3.1 after a 2-hr release into HU (Quench-HU-Pulse). Cells were analyzed by fluorescence microscopy, and fluorescence intensity per nucleus was quantified using ImageJ. (D) Stable HeLa cells expressing WT H3.1-GFP or a K4A mutant (clone D1) were left untreated or exposed to MMC for 24 hr and analyzed by FRAP. (E) WT DT40 cells or FANCD2 −/− cells expressing the indicated GFP-tagged chFANCD2 variants were treated as described in Figure 3 . Arrows indicate mean ratios. (F) Stable U-2-OS-GFP-RAD51 cells were co-transfected with the indicated siRNA and plasmids encoding either WT or chaperone-dead (R302W) mCherry-FANCD2 and exposed to MMC, and the mobility of GFP-RAD51 was analyzed by FRAP. (G) Model for the role of SET1A-dependent H3K4 methylation in protecting stalled replication forks. Upon fork reversal during replication stress, H3K4me1 by SETD1A acts to restrict CHD4 localization and promote FANCD2-dependent histone mobility. This chaperone activity is vital to stabilize RAD51 nucleofilaments, preventing nucleolytic degradation of stalled forks by DNA2. The plots in (D) and (F) represent mean ± SD relative fluorescence intensities from n = 21–26 and 25–48 cells, respectively. ∗ p

    Techniques Used: Transfection, Expressing, Mutagenesis, Fluorescence, Microscopy, Methylation, Activity Assay

    4) Product Images from "CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans"

    Article Title: CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0246-x

    Subcellular localization of CaGdt1 and its function in virulence. a Functional test of the GDT1-GFP allele in the sensitivity of C. albicans cells to Congo red (CR), Calcofluor white (CFW) and cyclosporine A (CsA) in WJCA102 cells ( pmr1/pmr1 gdt1/GDT1-GFP ). b co-localization of CaGDT1-GFP and Golgi-Tracker Red marker in the WJCA102 cells. c Co-localization of CaGDT1-GFP and CaPMR1-HA in WJCA111 cells through indirect immunofluorescent approach. Images of differential interference contrast (DIC), GFP, red florescence derived from Golgi-Track Red dye ( b ) or from goat anti-mouse IgG conjugated to Alexa Fluor 555 (for CaPMR1-HA protein) ( c ) and their merged images are presented. Scale bar, 5 μm
    Figure Legend Snippet: Subcellular localization of CaGdt1 and its function in virulence. a Functional test of the GDT1-GFP allele in the sensitivity of C. albicans cells to Congo red (CR), Calcofluor white (CFW) and cyclosporine A (CsA) in WJCA102 cells ( pmr1/pmr1 gdt1/GDT1-GFP ). b co-localization of CaGDT1-GFP and Golgi-Tracker Red marker in the WJCA102 cells. c Co-localization of CaGDT1-GFP and CaPMR1-HA in WJCA111 cells through indirect immunofluorescent approach. Images of differential interference contrast (DIC), GFP, red florescence derived from Golgi-Track Red dye ( b ) or from goat anti-mouse IgG conjugated to Alexa Fluor 555 (for CaPMR1-HA protein) ( c ) and their merged images are presented. Scale bar, 5 μm

    Techniques Used: Functional Assay, Marker, Derivative Assay

    5) Product Images from "Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors"

    Article Title: Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.01.011

    Generation of Human iTP Cells from Pancreatic Tissue (A) The morphologies of human pancreatic tissue, GTE cells, iPSCs, and iTP cells. Scale bar, 200 μm. (B) Numbers of colonies of iTP and iPSCs. Episomal plasmid vectors were transfected into human pancreatic tissue, and the number of colonies was counted after 30–45 days. (C) qRT-PCR analysis of PDX1, a marker of pancreatic stem/progenitor cells, in iTP and iPSCs. Eight iTP clones and two iPS clones were evaluated for PDX1 expression using qRT-PCR. The data are expressed as the PDX1-to-GAPDH ratio, with the ratio of pancreatic tissue arbitrarily set to 1 (n = 5). Error bars represent the SE. (D) Copy numbers of episomal plasmid vectors in iTP and iPS clones. Pancreatic tissue 6 days after electroporation of plasmid vectors expressing six reprogramming factors were analyzed (Pa-d6) as a positive control.
    Figure Legend Snippet: Generation of Human iTP Cells from Pancreatic Tissue (A) The morphologies of human pancreatic tissue, GTE cells, iPSCs, and iTP cells. Scale bar, 200 μm. (B) Numbers of colonies of iTP and iPSCs. Episomal plasmid vectors were transfected into human pancreatic tissue, and the number of colonies was counted after 30–45 days. (C) qRT-PCR analysis of PDX1, a marker of pancreatic stem/progenitor cells, in iTP and iPSCs. Eight iTP clones and two iPS clones were evaluated for PDX1 expression using qRT-PCR. The data are expressed as the PDX1-to-GAPDH ratio, with the ratio of pancreatic tissue arbitrarily set to 1 (n = 5). Error bars represent the SE. (D) Copy numbers of episomal plasmid vectors in iTP and iPS clones. Pancreatic tissue 6 days after electroporation of plasmid vectors expressing six reprogramming factors were analyzed (Pa-d6) as a positive control.

    Techniques Used: Plasmid Preparation, Transfection, Quantitative RT-PCR, Marker, Clone Assay, Expressing, Electroporation, Positive Control

    6) Product Images from "Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes"

    Article Title: Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes

    Journal: Cytoskeleton (Hoboken, N.j.)

    doi: 10.1002/cm.20419

    Myosin IIA and - IIB knock-downs have distinct effects on PMA-induced macropinocytosis. (A) Neuro-2a cells were transfected with non-silencing control (CTL) or siRNA targeting myosin IIA (IIA1 and IIA2) or myosin II B (IIB1 and IIB2). FITC-dextran was added with PMA for 2 minutes to assay the formation of macropinoctyosis and the percentage of cells containing dex+ macropinosomes were quantified. (B) Myosin IIB knockdown cells (IIB1 KD and IIB2 KD) show a significant decrease in dex+ cells whereas myosin IIA-knockdown cells (IIA1 KD and IIA2 KD) show a significant increase in dextran uptake. (C) Horseradish peroxidase (HRP) was also used to measure macropinosome formation in the cells transfected with CTL, or IIA1 or IIB1 or co-transfected with both IIA1 and IIB1. Data were normalized to the level of HRP uptake of the unstimulated control cells included in each experiment. HRP uptake was significantly increased in IIA1 KD cells but reduced in IIB1 KD or IIA1/IIB1 double knockdown cells. Scale bar=10 μm; scale bar in inset=1 μm.
    Figure Legend Snippet: Myosin IIA and - IIB knock-downs have distinct effects on PMA-induced macropinocytosis. (A) Neuro-2a cells were transfected with non-silencing control (CTL) or siRNA targeting myosin IIA (IIA1 and IIA2) or myosin II B (IIB1 and IIB2). FITC-dextran was added with PMA for 2 minutes to assay the formation of macropinoctyosis and the percentage of cells containing dex+ macropinosomes were quantified. (B) Myosin IIB knockdown cells (IIB1 KD and IIB2 KD) show a significant decrease in dex+ cells whereas myosin IIA-knockdown cells (IIA1 KD and IIA2 KD) show a significant increase in dextran uptake. (C) Horseradish peroxidase (HRP) was also used to measure macropinosome formation in the cells transfected with CTL, or IIA1 or IIB1 or co-transfected with both IIA1 and IIB1. Data were normalized to the level of HRP uptake of the unstimulated control cells included in each experiment. HRP uptake was significantly increased in IIA1 KD cells but reduced in IIB1 KD or IIA1/IIB1 double knockdown cells. Scale bar=10 μm; scale bar in inset=1 μm.

    Techniques Used: Transfection, CTL Assay

    Knock-down of myosin IIA and IIB showed opposite effects on macropinocytosis induced by IGF-1. (A) IGF-1 rapidly induced macropinocytosis in the Neuro-2a cells, more around the cell bodies than on the neurites. Myosin IIB appeared to localize in the areas of membrane ruffles whereas myosin IIA was more concentrated around the nucleus and neurites. (B) HRP uptake was performed on the cells transfected with CTL, IIA1, IIB1 or cotransfected with IIA1 and IIB1 siRNAs. IIB1 KD as well as IIA1+IIB1 KD cells showed diminished HRP uptake. In contrast, IIA1 KD cells exhibited a significant increase in HRP uptake. Scale bar=10 μm; 4× magnification shown in inset.
    Figure Legend Snippet: Knock-down of myosin IIA and IIB showed opposite effects on macropinocytosis induced by IGF-1. (A) IGF-1 rapidly induced macropinocytosis in the Neuro-2a cells, more around the cell bodies than on the neurites. Myosin IIB appeared to localize in the areas of membrane ruffles whereas myosin IIA was more concentrated around the nucleus and neurites. (B) HRP uptake was performed on the cells transfected with CTL, IIA1, IIB1 or cotransfected with IIA1 and IIB1 siRNAs. IIB1 KD as well as IIA1+IIB1 KD cells showed diminished HRP uptake. In contrast, IIA1 KD cells exhibited a significant increase in HRP uptake. Scale bar=10 μm; 4× magnification shown in inset.

    Techniques Used: Transfection, CTL Assay

    7) Product Images from "VHL-dependent regulation of a ?-dystroglycan glycoform and glycogene expression in renal cancer"

    Article Title: VHL-dependent regulation of a ?-dystroglycan glycoform and glycogene expression in renal cancer

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2013.2066

    Different forms of β-dystroglycan arise due to differential glycosylation. UMRC2−/+VHL lysates were deglycosylated (A) using PNGase F to remove N-linked glycans and (B) using a cocktail of enzymes to remove N- and O-linked glycans (for details of these enzymes see Table I ). Mock digests were carried out without enzyme(s). Changes in the gel mobility of β-dystroglycan were measured using immunoblotting. (C) Deglycosylation of bovine fetuin (as measured by SDS PAGE and silver staining) was monitored as an external control; a representative example is shown.
    Figure Legend Snippet: Different forms of β-dystroglycan arise due to differential glycosylation. UMRC2−/+VHL lysates were deglycosylated (A) using PNGase F to remove N-linked glycans and (B) using a cocktail of enzymes to remove N- and O-linked glycans (for details of these enzymes see Table I ). Mock digests were carried out without enzyme(s). Changes in the gel mobility of β-dystroglycan were measured using immunoblotting. (C) Deglycosylation of bovine fetuin (as measured by SDS PAGE and silver staining) was monitored as an external control; a representative example is shown.

    Techniques Used: SDS Page, Silver Staining

    8) Product Images from "MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice"

    Article Title: MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003280

    Western blot analysis of MMR and DHFR protein levels. MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods . B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.
    Figure Legend Snippet: Western blot analysis of MMR and DHFR protein levels. MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods . B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.

    Techniques Used: Western Blot, Expressing, Mouse Assay

    9) Product Images from "Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity"

    Article Title: Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085401

    Effects of immunization with SmCB or FhCL without adjuvant on immune responses, assessed 6 days post infection. (a) Titres of peptidase specific IgG were measured in the sera of immunized mice by ELISA. The data shown is representative of 3 independent experiments and displays the inverse of the end point serum dilution. (b) The isotype of peptidase specific antibodies in the sera of mice was determined by ELISA. The data shown is representative of 5 independent experiments. Each column represents the delta mean absorbance ± the standard error around the mean (SD). (c) Spleens were harvested from mice that were infected following immunization with SmCB1 or a combination of SmCB1 and FhCL1. The quantity of cytokine (IL-4, IL-5, IL-13 and IFN-γ) released into culture media by spleen cells stimulated with SmCB1 in vitro was measured by ELISA. Supernatants from cells incubated in media only served as negative controls. Each column represents the mean ± SD.
    Figure Legend Snippet: Effects of immunization with SmCB or FhCL without adjuvant on immune responses, assessed 6 days post infection. (a) Titres of peptidase specific IgG were measured in the sera of immunized mice by ELISA. The data shown is representative of 3 independent experiments and displays the inverse of the end point serum dilution. (b) The isotype of peptidase specific antibodies in the sera of mice was determined by ELISA. The data shown is representative of 5 independent experiments. Each column represents the delta mean absorbance ± the standard error around the mean (SD). (c) Spleens were harvested from mice that were infected following immunization with SmCB1 or a combination of SmCB1 and FhCL1. The quantity of cytokine (IL-4, IL-5, IL-13 and IFN-γ) released into culture media by spleen cells stimulated with SmCB1 in vitro was measured by ELISA. Supernatants from cells incubated in media only served as negative controls. Each column represents the mean ± SD.

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Incubation

    10) Product Images from "Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance *"

    Article Title: Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.463372

    Culture of hESCs on feeder cell-derived ECM. A , phase-contrast images of ECMs isolated from CD1 MEFs at P4 and P9 and from ihPSFs by alkali/detergent extraction. Scale bars , 100 μm. B , phase-contrast images showing morphology of HUES1 cells cultured for five consecutive passages on ECM derived from CD1 P4 and P9, ihPSF, and hPSF. Scale bars , 100 μm. C , HUES1 cells cultured on CD1 P4 and P9 ECM stained positively for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81 after multiple (up to four) passages. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm. D , HUES1 cells cultured on ihPSF-derived ECM stained positively for pluripotency-associated markers Nanog, Oct4, and Sox2 after multiple (up to four) passages, whereas HUES1 cells cultured on hPSF-derived ECM lost the expression of pluripotency-associated markers Nanog, Oct4, and Sox2. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 50 μm.
    Figure Legend Snippet: Culture of hESCs on feeder cell-derived ECM. A , phase-contrast images of ECMs isolated from CD1 MEFs at P4 and P9 and from ihPSFs by alkali/detergent extraction. Scale bars , 100 μm. B , phase-contrast images showing morphology of HUES1 cells cultured for five consecutive passages on ECM derived from CD1 P4 and P9, ihPSF, and hPSF. Scale bars , 100 μm. C , HUES1 cells cultured on CD1 P4 and P9 ECM stained positively for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81 after multiple (up to four) passages. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm. D , HUES1 cells cultured on ihPSF-derived ECM stained positively for pluripotency-associated markers Nanog, Oct4, and Sox2 after multiple (up to four) passages, whereas HUES1 cells cultured on hPSF-derived ECM lost the expression of pluripotency-associated markers Nanog, Oct4, and Sox2. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 50 μm.

    Techniques Used: Derivative Assay, Isolation, Cell Culture, Staining, Marker, Expressing

    Culture of hESCs on single ECM substrates or on substrates in combination with 5 μg/ml fibronectin. A , HUES1 cells were successfully cultured over three passages on fibrillin-1 coated at two different concentrations (10 and 20 μg/ml), similar to feeder-free culture on fibronectin (50 μg/ml). B , fibulin-2 and perlecan supported HUES1 cell culture only in combination with 5 μg/ml fibronectin; on these substrates, hESCs were maintained over three passages. C, HUES1 and HUES7 cells were successfully cultured over five passages on fibrillin-1 (10 μg/ml) and were positive for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm.
    Figure Legend Snippet: Culture of hESCs on single ECM substrates or on substrates in combination with 5 μg/ml fibronectin. A , HUES1 cells were successfully cultured over three passages on fibrillin-1 coated at two different concentrations (10 and 20 μg/ml), similar to feeder-free culture on fibronectin (50 μg/ml). B , fibulin-2 and perlecan supported HUES1 cell culture only in combination with 5 μg/ml fibronectin; on these substrates, hESCs were maintained over three passages. C, HUES1 and HUES7 cells were successfully cultured over five passages on fibrillin-1 (10 μg/ml) and were positive for pluripotency-associated markers Nanog and Oct4 and surface marker TRA-1-81. Cell nuclei were stained with DAPI ( blue ; insets ). Scale bars , 100 μm.

    Techniques Used: Cell Culture, Marker, Staining

    Interaction network analysis of human feeder cell- and hESC-derived ECM. A–C , proteins classified as extracellular or cell surface were converted to protein-protein interaction network models. Interaction networks were clustered using the yFiles Organic algorithm. Topological parameters were computed for ihPSF ( A ), hPSF ( B ), and HUES1 ( C ) ECM interaction networks as described under “Experimental Procedures.” Proteins identified by MS (nodes) are colored according to their clustering coefficient in each interaction network. Node diameter is proportional to number of connected neighbors (degree). Insets show the most highly connected region of each interaction network in detail ( A and B ); for clarity, nodes are labeled with gene names. Disconnected nodes are not displayed. Proteins classified as extracellular are displayed as circular nodes ; proteins classified as cell surface are displayed as rectangular nodes . Proteins detected in both ihPSF and hPSF ECMs are indicated by a black node border ; a gray node border indicates unique identification in ECM derived from that cell type ( A and B ). Fibronectin ( FN1 ), which was used as a substrate to culture HUES1 cells in the absence of feeders, is indicated by a dashed node border ( C ).
    Figure Legend Snippet: Interaction network analysis of human feeder cell- and hESC-derived ECM. A–C , proteins classified as extracellular or cell surface were converted to protein-protein interaction network models. Interaction networks were clustered using the yFiles Organic algorithm. Topological parameters were computed for ihPSF ( A ), hPSF ( B ), and HUES1 ( C ) ECM interaction networks as described under “Experimental Procedures.” Proteins identified by MS (nodes) are colored according to their clustering coefficient in each interaction network. Node diameter is proportional to number of connected neighbors (degree). Insets show the most highly connected region of each interaction network in detail ( A and B ); for clarity, nodes are labeled with gene names. Disconnected nodes are not displayed. Proteins classified as extracellular are displayed as circular nodes ; proteins classified as cell surface are displayed as rectangular nodes . Proteins detected in both ihPSF and hPSF ECMs are indicated by a black node border ; a gray node border indicates unique identification in ECM derived from that cell type ( A and B ). Fibronectin ( FN1 ), which was used as a substrate to culture HUES1 cells in the absence of feeders, is indicated by a dashed node border ( C ).

    Techniques Used: Derivative Assay, Mass Spectrometry, Labeling

    11) Product Images from "Csn3 Gene Is Regulated by All-Trans Retinoic Acid during Neural Differentiation in Mouse P19 Cells"

    Article Title: Csn3 Gene Is Regulated by All-Trans Retinoic Acid during Neural Differentiation in Mouse P19 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061938

    Expression of Csn3 in mouse ES cells and in mouse embryonic development. ( A ) Time course RT-PCR analysis of Csn3 mRNA expression in mouse ES cells (EB5). Total RNA was extracted from EB5 cells treated with ATRA (upper panel) or DMSO vehicle (lower panel) after 0, 3, 6, 12, 24, 48, 72 or 96 h. RT-PCR was performed using gene-specific primers as described in Materials and Methods. Expression of Oct3/4 and Mash1 were also examined to show the differentiating status. Gapdh was used as a loading control. PCR products were then subjected to electrophoresis through a 1.5% agarose gel and stained with ethidium bromide. Numbers in parentheses next to the gene symbols indicate the number of PCR cycles. ( B ) RT-PCR analysis of Csn3 mRNA expression in mouse embryos at different developmental stages (E7–E17). The amplified PCR products from the mouse embryos were resolved in 1.5% agarose gel and stained with ethidium bromide. Gapdh was used as a loading control.
    Figure Legend Snippet: Expression of Csn3 in mouse ES cells and in mouse embryonic development. ( A ) Time course RT-PCR analysis of Csn3 mRNA expression in mouse ES cells (EB5). Total RNA was extracted from EB5 cells treated with ATRA (upper panel) or DMSO vehicle (lower panel) after 0, 3, 6, 12, 24, 48, 72 or 96 h. RT-PCR was performed using gene-specific primers as described in Materials and Methods. Expression of Oct3/4 and Mash1 were also examined to show the differentiating status. Gapdh was used as a loading control. PCR products were then subjected to electrophoresis through a 1.5% agarose gel and stained with ethidium bromide. Numbers in parentheses next to the gene symbols indicate the number of PCR cycles. ( B ) RT-PCR analysis of Csn3 mRNA expression in mouse embryos at different developmental stages (E7–E17). The amplified PCR products from the mouse embryos were resolved in 1.5% agarose gel and stained with ethidium bromide. Gapdh was used as a loading control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Staining, Amplification

    12) Product Images from "MicroRNAs Are Indispensable for Reprogramming Mouse Embryonic Fibroblasts into Induced Stem Cell-Like Cells"

    Article Title: MicroRNAs Are Indispensable for Reprogramming Mouse Embryonic Fibroblasts into Induced Stem Cell-Like Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039239

    Dicer -null MEFs lacking miRNAs fail to reprogram. ( A, B ) Timelines of attempt at reprogramming Dicer -null MEFs. The main difference between the two strategies is that ( A ) transduces reprogramming transcription factors (TFs) 6 days post induction with Cre, while ( B ) transduces TFs 1 day post induction with Cre. Dicer -null MEFs could not be reprogrammed when reprogramming factors were transduced 6 days after induction. However, reprogramming Dicer -null MEFs was possible when reprogramming factors were transduced 1 day post induction with Cre. ( C ) Dicer +/+ , Dicer Δ/+ , and Dicer f/f MEFs consistently reprogrammed into iPSCs with reprogramming factors. These iPSCs stained for alkaline phosphatase. Dicer Δ/Δ MEFs reprogrammed to form induced stem cell-like cell colonies that stained for alkaline phosphatase when reprogramming factors were transduced 1 day post induction with Cre (Δ/Δ-1dpi). Transducing reprogramming factors 6 dpi (Δ/Δ-6dpi) or not transducing any factors (No TF) never reprogrammed Dicer -null MEFs. Boxed areas represent magnified view. ( D ) Genomic PCR confirmed induced stem cell-like cell colonies formed by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) having Dicer Δ/Δ genotype (floxed DNA band). Control iPSC colonies (f/f iPSC) formed without Cre induction had Dicer f/f genotype (flox DNA band). ( E ) Residual Dicer protein is still present 1 day after deletion of Dicer gene. By 6 days post induction (dpi) with Cre, residual Dicer protein is completely degraded, inhibiting cellular reprogramming. ( F, G ) Wild-type ESCs, Dicer f/f iPSCs (f/f iPSC), and Dicer Δ/Δ induced stem cell-like cells generated by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) expressed all stem cell markers tested by RT-PCR ( F ), and immunofluorescence ( G ). ( H ) Dicer f/f iPSCs and Dicer Δ/Δ induced stem cell-like cells acquired ESC methylation patterns in Oct4 and Nanog promoters.
    Figure Legend Snippet: Dicer -null MEFs lacking miRNAs fail to reprogram. ( A, B ) Timelines of attempt at reprogramming Dicer -null MEFs. The main difference between the two strategies is that ( A ) transduces reprogramming transcription factors (TFs) 6 days post induction with Cre, while ( B ) transduces TFs 1 day post induction with Cre. Dicer -null MEFs could not be reprogrammed when reprogramming factors were transduced 6 days after induction. However, reprogramming Dicer -null MEFs was possible when reprogramming factors were transduced 1 day post induction with Cre. ( C ) Dicer +/+ , Dicer Δ/+ , and Dicer f/f MEFs consistently reprogrammed into iPSCs with reprogramming factors. These iPSCs stained for alkaline phosphatase. Dicer Δ/Δ MEFs reprogrammed to form induced stem cell-like cell colonies that stained for alkaline phosphatase when reprogramming factors were transduced 1 day post induction with Cre (Δ/Δ-1dpi). Transducing reprogramming factors 6 dpi (Δ/Δ-6dpi) or not transducing any factors (No TF) never reprogrammed Dicer -null MEFs. Boxed areas represent magnified view. ( D ) Genomic PCR confirmed induced stem cell-like cell colonies formed by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) having Dicer Δ/Δ genotype (floxed DNA band). Control iPSC colonies (f/f iPSC) formed without Cre induction had Dicer f/f genotype (flox DNA band). ( E ) Residual Dicer protein is still present 1 day after deletion of Dicer gene. By 6 days post induction (dpi) with Cre, residual Dicer protein is completely degraded, inhibiting cellular reprogramming. ( F, G ) Wild-type ESCs, Dicer f/f iPSCs (f/f iPSC), and Dicer Δ/Δ induced stem cell-like cells generated by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) expressed all stem cell markers tested by RT-PCR ( F ), and immunofluorescence ( G ). ( H ) Dicer f/f iPSCs and Dicer Δ/Δ induced stem cell-like cells acquired ESC methylation patterns in Oct4 and Nanog promoters.

    Techniques Used: Staining, Polymerase Chain Reaction, Generated, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Methylation

    Human Dicer expression in Dicer -null MEFs allows generation of iPSCs. ( A ) Timeline of reprogramming Dicer -null MEFs rescued with human Dicer . Once human Dicer cDNA integrated into the Dicer Δ/Δ MEF genome, reprogramming became possible even when factors were transduced 6 days after Cre induction. ( B ) Rescued iPSCs (ResDcr iPSC) lacked mouse Dicer ( mDcr ), but instead expressed human Dicer ( hDcr ) gene, verified by RT-PCR. ( C, D, E ) Dicer Δ/Δ MEFs expressing human Dicer can reprogram to become iPSCs. Rescued iPSCs expressing human Dicer showed ESC morphology and stained for alkaline phosphatase (boxed areas represent magnified view) ( C ), and expressed stem cell markers tested by RT-PCR ( D ) and immunofluorescence ( E ). ( F ) Rescued iPSC promoters for stem cell genes Oct4 and Nanog became demethylated, resembling wild-type ESCs. ( G ) Upon subcutaneous injection into SCID mice, rescued iPSCs formed teratomas that showed differentiation into all three germ layers. ( H ) Human Dicer can cleave mouse pre-miRNAs into mature miRNAs. qPCR for a panel of mature miRNAs in rescued iPSCs (ResDcr) lacking mouse Dicer demonstrated comparable expression levels to that of wild-type ESCs (W4). In contrast, mature miRNAs were completely depleted in Dicer -null ESCs (Dicer Δ/Δ ). Each value is represented relative to an assigned W4 value of 1.0 for that miRNA. Data are presented as mean +/− SD.
    Figure Legend Snippet: Human Dicer expression in Dicer -null MEFs allows generation of iPSCs. ( A ) Timeline of reprogramming Dicer -null MEFs rescued with human Dicer . Once human Dicer cDNA integrated into the Dicer Δ/Δ MEF genome, reprogramming became possible even when factors were transduced 6 days after Cre induction. ( B ) Rescued iPSCs (ResDcr iPSC) lacked mouse Dicer ( mDcr ), but instead expressed human Dicer ( hDcr ) gene, verified by RT-PCR. ( C, D, E ) Dicer Δ/Δ MEFs expressing human Dicer can reprogram to become iPSCs. Rescued iPSCs expressing human Dicer showed ESC morphology and stained for alkaline phosphatase (boxed areas represent magnified view) ( C ), and expressed stem cell markers tested by RT-PCR ( D ) and immunofluorescence ( E ). ( F ) Rescued iPSC promoters for stem cell genes Oct4 and Nanog became demethylated, resembling wild-type ESCs. ( G ) Upon subcutaneous injection into SCID mice, rescued iPSCs formed teratomas that showed differentiation into all three germ layers. ( H ) Human Dicer can cleave mouse pre-miRNAs into mature miRNAs. qPCR for a panel of mature miRNAs in rescued iPSCs (ResDcr) lacking mouse Dicer demonstrated comparable expression levels to that of wild-type ESCs (W4). In contrast, mature miRNAs were completely depleted in Dicer -null ESCs (Dicer Δ/Δ ). Each value is represented relative to an assigned W4 value of 1.0 for that miRNA. Data are presented as mean +/− SD.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Immunofluorescence, Injection, Mouse Assay, Real-time Polymerase Chain Reaction

    13) Product Images from "Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia"

    Article Title: Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia

    Journal: Oncotarget

    doi:

    Schematic diagram of adenoviral constructs and CRAd infectivity in leukemic cells (A) Compared to wild-type Ad5, SG511-BECN contains a human telomerase reverse transcriptase (hTERT) promoter-controlled E1 expression cassette in forward orientation, an E1B 55-kDa deletion, and Beclin-1 transgene in E3 region. The fiber was modified by an Ad5/11 chimeric fiber. (B) Kusumi-1 and K562 cells were infected with 50 MOI of SG511-GFP for 24 h and then observed under a fluorescence microscope (200×) (top panel). K562 cells were treated with the indicated amount of the virus for 24 h and then collected for analysis by FACS (bottom panel). (C) Bone marrow cells obtained from a patient with AML were infected with SG511-GFP at an MOI of 50, and then cultured for 5 days in colony culture assay. Photographs were viewed under a light microscope and fluorescence microscope, respectively (400×). (D) Beclin-1 and E1A expression was determined by Western blotting of K562 cells infected with SG511-BECN at the indicated concentrations for 48 h. Actin was Western blotted for equal loading. (E) Following SG511-BECN treatment for 48 h, Beclin-1 (green) and ER (red) in K562 cells were detected by confocal immunofluorescence microscopy. Representative data of three experiments is shown.
    Figure Legend Snippet: Schematic diagram of adenoviral constructs and CRAd infectivity in leukemic cells (A) Compared to wild-type Ad5, SG511-BECN contains a human telomerase reverse transcriptase (hTERT) promoter-controlled E1 expression cassette in forward orientation, an E1B 55-kDa deletion, and Beclin-1 transgene in E3 region. The fiber was modified by an Ad5/11 chimeric fiber. (B) Kusumi-1 and K562 cells were infected with 50 MOI of SG511-GFP for 24 h and then observed under a fluorescence microscope (200×) (top panel). K562 cells were treated with the indicated amount of the virus for 24 h and then collected for analysis by FACS (bottom panel). (C) Bone marrow cells obtained from a patient with AML were infected with SG511-GFP at an MOI of 50, and then cultured for 5 days in colony culture assay. Photographs were viewed under a light microscope and fluorescence microscope, respectively (400×). (D) Beclin-1 and E1A expression was determined by Western blotting of K562 cells infected with SG511-BECN at the indicated concentrations for 48 h. Actin was Western blotted for equal loading. (E) Following SG511-BECN treatment for 48 h, Beclin-1 (green) and ER (red) in K562 cells were detected by confocal immunofluorescence microscopy. Representative data of three experiments is shown.

    Techniques Used: Construct, Infection, Expressing, Modification, Fluorescence, Microscopy, FACS, Cell Culture, Light Microscopy, Western Blot, Immunofluorescence

    14) Product Images from "Stauprimide Priming of Human Embryonic Stem Cells toward Definitive Endoderm"

    Article Title: Stauprimide Priming of Human Embryonic Stem Cells toward Definitive Endoderm

    Journal: Cell Journal (Yakhteh)

    doi:

    Gene and protein expression analysis of definitive endoderm (DE) markers in experimental and control groups at day 4. A. Diagrammatic representation of the experimental groups (Spd-A50/A25 and Spd-IDE1/2), positive control (W/A100-A100) and negative control (DMSO) for endoderm induction of human embryonic stem cells (hESCs). B. Lineage-specific gene expression analysis of Spd-A50-treated Royan H6 human embryonic stem cells (hESC-RH6). In control groups, hESCs were treated with 0.1% dimethyl sulfoxide (DMSO) for 4 days and considered as the negative control. Cells were treated with Wnt3a and activin A for the first day, followed by treatment with activin A for the next three days (W/A100-A100) as the positive control. As determined by Q-PCR, the DE markers SOX17, FOXA2 and CXCR4 highly expressed in Spd-A50-treated Royan H6 hESCs while SOX7 [visceral endoderm (VE) marker], SOX1 (neuroectoderm marker) and OCT4 (pluripotency marker) had low levels of expression. The target gene expression level was normalized to GAPDH and presented relative to hESC. Data are presented as mean ± SD. C. Immunofluorescent staining of Spd-A50-treated human embryonic stem cells (hESCs) showed SOX17+ (green) populations comparable to that of W/A100-A100- (positive control) cells. DAPI; 4=,6-diamidino-2-phenylindole.
    Figure Legend Snippet: Gene and protein expression analysis of definitive endoderm (DE) markers in experimental and control groups at day 4. A. Diagrammatic representation of the experimental groups (Spd-A50/A25 and Spd-IDE1/2), positive control (W/A100-A100) and negative control (DMSO) for endoderm induction of human embryonic stem cells (hESCs). B. Lineage-specific gene expression analysis of Spd-A50-treated Royan H6 human embryonic stem cells (hESC-RH6). In control groups, hESCs were treated with 0.1% dimethyl sulfoxide (DMSO) for 4 days and considered as the negative control. Cells were treated with Wnt3a and activin A for the first day, followed by treatment with activin A for the next three days (W/A100-A100) as the positive control. As determined by Q-PCR, the DE markers SOX17, FOXA2 and CXCR4 highly expressed in Spd-A50-treated Royan H6 hESCs while SOX7 [visceral endoderm (VE) marker], SOX1 (neuroectoderm marker) and OCT4 (pluripotency marker) had low levels of expression. The target gene expression level was normalized to GAPDH and presented relative to hESC. Data are presented as mean ± SD. C. Immunofluorescent staining of Spd-A50-treated human embryonic stem cells (hESCs) showed SOX17+ (green) populations comparable to that of W/A100-A100- (positive control) cells. DAPI; 4=,6-diamidino-2-phenylindole.

    Techniques Used: Expressing, Positive Control, Negative Control, Polymerase Chain Reaction, Marker, Staining

    15) Product Images from "Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells"

    Article Title: Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096279

    Muscle marker expression in differentiating mdx iPSC. W9 and W987 iPSC and ESC were differentiated in vitro , and at various time points, cells were stained with the SM/C-2.6 antibody and analyzed by flow cytometry (FC). (a) FC plots of W9, W987, and ESC differentiated for 13 days, indicating that approximately 4.5%, 46%, and 59% of cells were positive for SM/C-2.6, respectively, at this time point. Anti-rat IgG was used as a staining control. (b) Differentiation efficiency at the day 6, 13, 20, 27, and 34 time points for W9, W987, and ESC, as judged by percent positive for SM/C-2.6. (c) qRT-PCR analysis of muscle lineage markers Pax7 , Pax3 , MyoD , and myogenin during the time course in W9, W987, and ESC. (d) qRT-PCR analysis of MyoD and myogenin expression in W987, W9 and ESC cells at 20 and 27 days of differentiation. (e) qRT-PCR analysis of dystrophin expression at various time points.
    Figure Legend Snippet: Muscle marker expression in differentiating mdx iPSC. W9 and W987 iPSC and ESC were differentiated in vitro , and at various time points, cells were stained with the SM/C-2.6 antibody and analyzed by flow cytometry (FC). (a) FC plots of W9, W987, and ESC differentiated for 13 days, indicating that approximately 4.5%, 46%, and 59% of cells were positive for SM/C-2.6, respectively, at this time point. Anti-rat IgG was used as a staining control. (b) Differentiation efficiency at the day 6, 13, 20, 27, and 34 time points for W9, W987, and ESC, as judged by percent positive for SM/C-2.6. (c) qRT-PCR analysis of muscle lineage markers Pax7 , Pax3 , MyoD , and myogenin during the time course in W9, W987, and ESC. (d) qRT-PCR analysis of MyoD and myogenin expression in W987, W9 and ESC cells at 20 and 27 days of differentiation. (e) qRT-PCR analysis of dystrophin expression at various time points.

    Techniques Used: Marker, Expressing, In Vitro, Staining, Flow Cytometry, Cytometry, Quantitative RT-PCR

    Myofiber differentiation and engraftment. (a) Immunofluorescence staining of dystrophin in W9, W987, and ESC. Myosin heavy chain (MHC) identified muscle cells after differentiation. DAPI was used to stain nuclei. (b) Myotube formation in differentiated W987 and W9 iPSC and ESC. Note that W9 iPSC did not form myotubes. (c) Engraftment of corrected mdx iPSC in mouse TA muscle. Approximately 750,000 W987 iPSC that were differentiated for 13 days in vitro and sorted for the SM/C-2.6 antibody were injected into the TA muscle of an irradiated mdx/;SCID mouse. After three weeks, muscle sections were prepared and stained. Staining for laminin delineated individual muscle fibers, while staining for dystrophin revealed engraftment of corrected iPSC (arrows). (d) Numbers of dystrophin-positive fibers per TA muscle, total section, are shown for engrafted muscle versus uninjected contralateral muscle (control).
    Figure Legend Snippet: Myofiber differentiation and engraftment. (a) Immunofluorescence staining of dystrophin in W9, W987, and ESC. Myosin heavy chain (MHC) identified muscle cells after differentiation. DAPI was used to stain nuclei. (b) Myotube formation in differentiated W987 and W9 iPSC and ESC. Note that W9 iPSC did not form myotubes. (c) Engraftment of corrected mdx iPSC in mouse TA muscle. Approximately 750,000 W987 iPSC that were differentiated for 13 days in vitro and sorted for the SM/C-2.6 antibody were injected into the TA muscle of an irradiated mdx/;SCID mouse. After three weeks, muscle sections were prepared and stained. Staining for laminin delineated individual muscle fibers, while staining for dystrophin revealed engraftment of corrected iPSC (arrows). (d) Numbers of dystrophin-positive fibers per TA muscle, total section, are shown for engrafted muscle versus uninjected contralateral muscle (control).

    Techniques Used: Immunofluorescence, Staining, In Vitro, Injection, Irradiation

    16) Product Images from "Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL"

    Article Title: Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-7-1

    Phosphorylation of Bad by Pim prevents Bcl-X L binding . A) GST-Bad was transfected into HeLa cells along with either empty pCMV5 vector or active (W) or kinase dead (D) FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed. GST pull downs were then performed on the extracts and imuunoblotted for Bcl-X L associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-X L , total Bad or FLAG. B) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cell were serum starved for 18 h and were then lysed. GST pull downs were then performed on the extracts and immunoblotted for Bcl-X L associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-X L, , total Bad and FLAG. C) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed. GST pull downs were then performed on the extracts and immunoblotted for Bcl-X L associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-X L , total Bad or FLAG.
    Figure Legend Snippet: Phosphorylation of Bad by Pim prevents Bcl-X L binding . A) GST-Bad was transfected into HeLa cells along with either empty pCMV5 vector or active (W) or kinase dead (D) FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed. GST pull downs were then performed on the extracts and imuunoblotted for Bcl-X L associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-X L , total Bad or FLAG. B) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cell were serum starved for 18 h and were then lysed. GST pull downs were then performed on the extracts and immunoblotted for Bcl-X L associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-X L, , total Bad and FLAG. C) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed. GST pull downs were then performed on the extracts and immunoblotted for Bcl-X L associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-X L , total Bad or FLAG.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Expressing, Construct

    Phosphorylation of Bad by Pim kinases in HEK-293 cells . A) FLAG-Pim-1, Pim-2 or Pim-3 were expressed in HEK-293 cells by transient transfection. 24 h after transfection cells were serum starved for 18 h and then stimulated with either IGF (100 ng/ml 5 min), sorbitol (0.5 M for 30 min), AICar (2 mM for 1 hour), UV-C (200Jm2 followed by 1 hour incubation at 37°C), hydrogen peroxide (1 mM for 1 hour), PMA (400 ng/ml for 15 min), anisomycin (10 μg/ml for 1 hour) or left unstimulated. Cells were lysed and Pim kinase activity measured by immunoprecipitation assays as described in the methods. B) GST-Bad was transfected into HEK-293 cells along with either empty pCMV5 vector or FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and then left unstimulated or stimulated with either PMA (400 ng/ml for 15 min) or forskolin (20 μM for 30 min). Cells were then lysed and extracts immunoblotted for Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad and FLAG (to monitor Pim-1, 2 or 3 expression). C) GST-Bad was transfected into HEK-293 cells with empty pCMV5 vector or active or kinase dead FLAG-Pim-1-3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad and FLAG (to monitor Pim-1, 2 or 3 expression).
    Figure Legend Snippet: Phosphorylation of Bad by Pim kinases in HEK-293 cells . A) FLAG-Pim-1, Pim-2 or Pim-3 were expressed in HEK-293 cells by transient transfection. 24 h after transfection cells were serum starved for 18 h and then stimulated with either IGF (100 ng/ml 5 min), sorbitol (0.5 M for 30 min), AICar (2 mM for 1 hour), UV-C (200Jm2 followed by 1 hour incubation at 37°C), hydrogen peroxide (1 mM for 1 hour), PMA (400 ng/ml for 15 min), anisomycin (10 μg/ml for 1 hour) or left unstimulated. Cells were lysed and Pim kinase activity measured by immunoprecipitation assays as described in the methods. B) GST-Bad was transfected into HEK-293 cells along with either empty pCMV5 vector or FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and then left unstimulated or stimulated with either PMA (400 ng/ml for 15 min) or forskolin (20 μM for 30 min). Cells were then lysed and extracts immunoblotted for Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad and FLAG (to monitor Pim-1, 2 or 3 expression). C) GST-Bad was transfected into HEK-293 cells with empty pCMV5 vector or active or kinase dead FLAG-Pim-1-3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad and FLAG (to monitor Pim-1, 2 or 3 expression).

    Techniques Used: Transfection, Incubation, Activity Assay, Immunoprecipitation, Plasmid Preparation, Expressing, Construct

    Ser136 is a critical site to allow phosphorylation of Bad by Pim in cells . A) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted with antibodies against Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad or FLAG. B) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed and extracts immunoblotted with antibodies against Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad or FLAG. C) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for total Bad or FLAG (to monitor expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. D) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed and extracts immunoblotted for total Bad or FLAG (to monitor expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. E) GST-Bad was transfected into HEK-293 cells along with either empty pCMV5 vector or active (W) or kinase dead (D) FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for total Bad or FLAG (to monitor Pim-1, 2 or 3 expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. F) As E except that GST pull downs were performed on the extracts and immunoblotted for 14-3-3 as described in the methods.
    Figure Legend Snippet: Ser136 is a critical site to allow phosphorylation of Bad by Pim in cells . A) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted with antibodies against Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad or FLAG. B) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed and extracts immunoblotted with antibodies against Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad or FLAG. C) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for total Bad or FLAG (to monitor expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. D) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed and extracts immunoblotted for total Bad or FLAG (to monitor expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. E) GST-Bad was transfected into HEK-293 cells along with either empty pCMV5 vector or active (W) or kinase dead (D) FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for total Bad or FLAG (to monitor Pim-1, 2 or 3 expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. F) As E except that GST pull downs were performed on the extracts and immunoblotted for 14-3-3 as described in the methods.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Construct

    17) Product Images from "Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters"

    Article Title: Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2009/848145

    Expression of epitope-tagged AUX1 in insect cells. (a) Sf9 cells at densities of 2 × 10 6 cells/mL were infected with recombinant baculoviruses expressing epitope-tagged AUX1 at a range of multiplicity of infections (MOIs) and harvested at 24–96 hours postinfection (hpi). Cell lysates (10 μ g) were resolved by SDS-PAGE and identified by western blotting with appropriate antibodies directed towards the epitope tags. Panels show data for a 72-hour postinfection only. Molecular weight (kDa) of marker proteins is denoted at the right-hand side of the figure. (b) Auxin binding to AUX1-containing membranes (72 hours postinfection) was assessed by a centrifugation-based radioisotope binding assay [ 12 ]. AUX1 displacement could be observed when membranes were incubated with 1 mM auxin analogues such as 2,4-D and 1-NOA but not when the unrelated weak acid benzoic acid (BA) was applied. (c) Solubilisation of HA-AUX1 from insect cell membranes. 100 μ g of membranes were incubated for 60 minutes at 4°C with detergents at greater than 2X critical micelle concentration. Solubilised material was separated from insoluble material by ultracentrifugation and equal percentages of the two fractions resolved by SDS-PAGE and immunoblotting. Detergent abbreviations: 3–16: Zwittergent 3–16 (Calbiochem); β -OG: β -octyl-glucoside; DM: n -decyl- β -D-maltoside decylmaltoside; DDM: n -dodecyl- β -D-maltoside.
    Figure Legend Snippet: Expression of epitope-tagged AUX1 in insect cells. (a) Sf9 cells at densities of 2 × 10 6 cells/mL were infected with recombinant baculoviruses expressing epitope-tagged AUX1 at a range of multiplicity of infections (MOIs) and harvested at 24–96 hours postinfection (hpi). Cell lysates (10 μ g) were resolved by SDS-PAGE and identified by western blotting with appropriate antibodies directed towards the epitope tags. Panels show data for a 72-hour postinfection only. Molecular weight (kDa) of marker proteins is denoted at the right-hand side of the figure. (b) Auxin binding to AUX1-containing membranes (72 hours postinfection) was assessed by a centrifugation-based radioisotope binding assay [ 12 ]. AUX1 displacement could be observed when membranes were incubated with 1 mM auxin analogues such as 2,4-D and 1-NOA but not when the unrelated weak acid benzoic acid (BA) was applied. (c) Solubilisation of HA-AUX1 from insect cell membranes. 100 μ g of membranes were incubated for 60 minutes at 4°C with detergents at greater than 2X critical micelle concentration. Solubilised material was separated from insoluble material by ultracentrifugation and equal percentages of the two fractions resolved by SDS-PAGE and immunoblotting. Detergent abbreviations: 3–16: Zwittergent 3–16 (Calbiochem); β -OG: β -octyl-glucoside; DM: n -decyl- β -D-maltoside decylmaltoside; DDM: n -dodecyl- β -D-maltoside.

    Techniques Used: Expressing, Infection, Recombinant, SDS Page, Western Blot, Molecular Weight, Marker, Binding Assay, Centrifugation, Incubation, Concentration Assay

    Blue native PAGE analysis of AUX1 expression in insect cells. N-HA-AUX1 membrane fractions from insect (Sf9) cells were solubilised in 0.1% (w/v) DDM in the presence of increasing concentrations of NaCl and were resolved by BN-PAGE on 6–16% gradient gels, transferred to PVDF and identified by immunoblotting with anti-HA antibodies. Molecular weight (kDa) of marker proteins is denoted at the left-hand side of the figure.
    Figure Legend Snippet: Blue native PAGE analysis of AUX1 expression in insect cells. N-HA-AUX1 membrane fractions from insect (Sf9) cells were solubilised in 0.1% (w/v) DDM in the presence of increasing concentrations of NaCl and were resolved by BN-PAGE on 6–16% gradient gels, transferred to PVDF and identified by immunoblotting with anti-HA antibodies. Molecular weight (kDa) of marker proteins is denoted at the left-hand side of the figure.

    Techniques Used: Blue Native PAGE, Expressing, Polyacrylamide Gel Electrophoresis, Molecular Weight, Marker

    Expression of epitope-tagged AUX1 in P. pastoris . P. pastoris strain KM71H transformed with plasmids encoding L2-His 6 3xFLAG-AUX1 or N-His 6 3xFLAG-AUX1 was grown, induced, and harvested as described in the Methods. Of the 32 cell lysates analysed by Western blotting (anti-FLAG antibody), only 6 are shown here for clarity, the other 26 showing no AUX1 expression. AUX1 was observed in a single N-His 6 3xFLAG-AUX1 culture (lane 5). A number of nonspecific bands reacting with the anti-FLAG antibody are denoted with asterisks, including one migrating just higher than the AUX1 band.
    Figure Legend Snippet: Expression of epitope-tagged AUX1 in P. pastoris . P. pastoris strain KM71H transformed with plasmids encoding L2-His 6 3xFLAG-AUX1 or N-His 6 3xFLAG-AUX1 was grown, induced, and harvested as described in the Methods. Of the 32 cell lysates analysed by Western blotting (anti-FLAG antibody), only 6 are shown here for clarity, the other 26 showing no AUX1 expression. AUX1 was observed in a single N-His 6 3xFLAG-AUX1 culture (lane 5). A number of nonspecific bands reacting with the anti-FLAG antibody are denoted with asterisks, including one migrating just higher than the AUX1 band.

    Techniques Used: Expressing, Transformation Assay, Western Blot

    Epitope-tagged forms of AUX1. Diagrammatic representation of AUX1 constructs. The predicted membrane topology of AUX1 is shown with TM helices represented as cylinders. The epitope sequences for the HIS 6 3xFLAG and HA tags are shown with the sites of insertion represented as triangles, with the specific residue number for the insertion site below.
    Figure Legend Snippet: Epitope-tagged forms of AUX1. Diagrammatic representation of AUX1 constructs. The predicted membrane topology of AUX1 is shown with TM helices represented as cylinders. The epitope sequences for the HIS 6 3xFLAG and HA tags are shown with the sites of insertion represented as triangles, with the specific residue number for the insertion site below.

    Techniques Used: Construct

    Expression of epitope-tagged AUX1 in mammalian cells. (a) HEK293T cells were transfected using polyethyleneimine with the indicated amounts of recombinant L2-His 6 3xFLAG or N-His 6 3xFLAG epitope-tagged AUX1 DNA (L2 and N, resp., below each lane), and harvested 72 hours posttransfection. (b) HEK293T cells were transfected using 8 μ g of either L2-His 6 3xFLAG-AUX1 or N-His 6 3xFLAG-AUX1 epitope-tagged AUX1 DNA and harvested at the indicated times posttransfection. Cells were lysed by sonication, and 10 μ g of lysates were resolved by SDS-PAGE and identified by western blotting with anti-FLAG antibodies. The asterisk identifies a nonspecific protein reacting with the anti-FLAG antibody. Molecular weight (kDa) of marker proteins is denoted at the right-hand side of the figure. (c) HEK293T cells were transfected on coverslips in 6-well dishes with cDNA encoding N-His 6 3XFLAG-AUX1 and were visualised 48 hours later by confocal microscopy after immunoblotting with an anti-FLAG primary antibody (1 : 100 dilution) and a GFP-conjugated secondary antibody (1 : 200). Cell nuclei were counter stained with DAPI. (d) Transport of [ 3 H]-IAA into U2OS cells transiently transfected with L2-His 6 3XFLAG-AUX1 compared to transport into cells transfected with empty vector. Data is expressed as a percentage of the transport rate into AUX1-transfected cells and represents the mean (± standard error) of 9 independent experiments with 2–4 determinations of transport in each transfection.
    Figure Legend Snippet: Expression of epitope-tagged AUX1 in mammalian cells. (a) HEK293T cells were transfected using polyethyleneimine with the indicated amounts of recombinant L2-His 6 3xFLAG or N-His 6 3xFLAG epitope-tagged AUX1 DNA (L2 and N, resp., below each lane), and harvested 72 hours posttransfection. (b) HEK293T cells were transfected using 8 μ g of either L2-His 6 3xFLAG-AUX1 or N-His 6 3xFLAG-AUX1 epitope-tagged AUX1 DNA and harvested at the indicated times posttransfection. Cells were lysed by sonication, and 10 μ g of lysates were resolved by SDS-PAGE and identified by western blotting with anti-FLAG antibodies. The asterisk identifies a nonspecific protein reacting with the anti-FLAG antibody. Molecular weight (kDa) of marker proteins is denoted at the right-hand side of the figure. (c) HEK293T cells were transfected on coverslips in 6-well dishes with cDNA encoding N-His 6 3XFLAG-AUX1 and were visualised 48 hours later by confocal microscopy after immunoblotting with an anti-FLAG primary antibody (1 : 100 dilution) and a GFP-conjugated secondary antibody (1 : 200). Cell nuclei were counter stained with DAPI. (d) Transport of [ 3 H]-IAA into U2OS cells transiently transfected with L2-His 6 3XFLAG-AUX1 compared to transport into cells transfected with empty vector. Data is expressed as a percentage of the transport rate into AUX1-transfected cells and represents the mean (± standard error) of 9 independent experiments with 2–4 determinations of transport in each transfection.

    Techniques Used: Expressing, Transfection, Recombinant, Sonication, SDS Page, Western Blot, Molecular Weight, Marker, Confocal Microscopy, Staining, Plasmid Preparation

    18) Product Images from "Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells"

    Article Title: Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-10-7

    Co-immunoprecipitation analyses to detect MAL2/MUC1 interactions in Triton X-100-soluble versus-insoluble fractions. MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation . Antisera employed in Western blot analyses are shown at the left, and sizes of detected proteins are shown at the right. (a) Fractions (Fr, as shown above the top panel) of 1 ml were collected, and aliquots from each were subjected to SDS-PAGE and Western blot analysis with MUC1 and MAL2 antisera. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. (b) Pooled fractions (fractions 1–4, which include lipid rafts, fractions 5–8 and fractions 9–12) were immunoprecipitated with MUC1 or MAL2 antisera either alone or with MAL2 peptides (+Pep), as shown at the top of the panel. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with MUC1 monoclonal antibody. Results shown represent those obtained from 3 independent experiments.
    Figure Legend Snippet: Co-immunoprecipitation analyses to detect MAL2/MUC1 interactions in Triton X-100-soluble versus-insoluble fractions. MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation . Antisera employed in Western blot analyses are shown at the left, and sizes of detected proteins are shown at the right. (a) Fractions (Fr, as shown above the top panel) of 1 ml were collected, and aliquots from each were subjected to SDS-PAGE and Western blot analysis with MUC1 and MAL2 antisera. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. (b) Pooled fractions (fractions 1–4, which include lipid rafts, fractions 5–8 and fractions 9–12) were immunoprecipitated with MUC1 or MAL2 antisera either alone or with MAL2 peptides (+Pep), as shown at the top of the panel. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with MUC1 monoclonal antibody. Results shown represent those obtained from 3 independent experiments.

    Techniques Used: Immunoprecipitation, Gradient Centrifugation, Western Blot, SDS Page

    Co-immunoprecipitation analyses verifying interactions between MAL2 and MUC1 . Total cell lysate from MCF-10A/Myc-MAL2 cells was immunoprecipitated with MUC1, c-Myc, or rabbit MAL2 antisera, either alone or with MAL2 peptides, as indicated above the top panel. Total cell lysate and immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analyses with antisera against MUC1, MAL2, c-Myc and D52, as indicated at the left. Sizes of detected proteins are indicated at the right. Results shown are representative of at least 3 independent experiments.
    Figure Legend Snippet: Co-immunoprecipitation analyses verifying interactions between MAL2 and MUC1 . Total cell lysate from MCF-10A/Myc-MAL2 cells was immunoprecipitated with MUC1, c-Myc, or rabbit MAL2 antisera, either alone or with MAL2 peptides, as indicated above the top panel. Total cell lysate and immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analyses with antisera against MUC1, MAL2, c-Myc and D52, as indicated at the left. Sizes of detected proteins are indicated at the right. Results shown are representative of at least 3 independent experiments.

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot

    19) Product Images from "Constitutive Activation of STAT5A Promotes Human Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation"

    Article Title: Constitutive Activation of STAT5A Promotes Human Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20041024

    Long-term cultures of CB CD34 + cells transduced with STAT5A(1*6) on MS5 stroma. (A) CB CD34 + cells were transduced with STAT5A(1*6) and plated on MS5 to generate CAFCs, which were harvested and replated onto new MS5 at day 10, weeks 3, 5, and 7 (not depicted), and weeks 10, 13, and 15 (indicated by the arrows). Cultures were weekly demidepopulated and cell counts of a representative experiment out of two independent experiments are shown. At week 14, nonadherent cells were used for FACS analysis (see Table I ), RT-PCR analysis (B), in which total RNA was isolated, and used in RT-PCR reactions with primers that amplify the STAT5A(1*6)-IRES2 cassette, cytospins (C), or CFC assays (D), in which cells were plated in methylcellulose.
    Figure Legend Snippet: Long-term cultures of CB CD34 + cells transduced with STAT5A(1*6) on MS5 stroma. (A) CB CD34 + cells were transduced with STAT5A(1*6) and plated on MS5 to generate CAFCs, which were harvested and replated onto new MS5 at day 10, weeks 3, 5, and 7 (not depicted), and weeks 10, 13, and 15 (indicated by the arrows). Cultures were weekly demidepopulated and cell counts of a representative experiment out of two independent experiments are shown. At week 14, nonadherent cells were used for FACS analysis (see Table I ), RT-PCR analysis (B), in which total RNA was isolated, and used in RT-PCR reactions with primers that amplify the STAT5A(1*6)-IRES2 cassette, cytospins (C), or CFC assays (D), in which cells were plated in methylcellulose.

    Techniques Used: Transduction, FACS, Reverse Transcription Polymerase Chain Reaction, Isolation

    STAT5A(1*6) induces early CAFCs in CB CD34 + cells. CB CD34 + cells were transduced with MiGR1 or STAT5A(1*6) as described in Fig. 1 and grown in cocultures on stromal cell lines as indicated. (A) Representative examples of CAFCs at day 10 observed in coculture studies with CB CD34 + cells transduced with STAT5A(1*6). (B) CAFC frequencies were determined on MS5 in limiting dilution conditions as described in Materials and Methods. (C) GFP + cells were sorted from the adherent population on MS5 at day 10 and used for FACS analysis and cytospins (D). (E) CB CD34 + cells were transduced with MiGR1 or STAT5A(1*6), after which the CD38 high and CD38 low populations were sorted and used to determine the CAFC frequencies on MS5 under limiting dilution conditions. First CAFC frequencies were determined at day 10, and cultures were either harvested and plated on new MS5 stroma to determine secondary CAFC frequencies (indicated in black as number of secondary CAFCs/number of primary CAFCs), or plated in methylcellulose to determine the number of CFCs/10 CAFCs (F). (G) CB CD34 + cells were transduced with MiGR1 or STAT5A(1*6) as described in Fig. 1 and plated for CFC assays in triplicate. After 2 wk, the colonies were scored and cells from each plate were plated for secondary CFCs. (H) Sublethally irradiated NOD-SCID recipients were injected with 2 × 10 5 GFP + day 10 STAT5A(1*6) CAFCs. Mice were killed at week 6 and the bone marrow was analyzed as indicated.
    Figure Legend Snippet: STAT5A(1*6) induces early CAFCs in CB CD34 + cells. CB CD34 + cells were transduced with MiGR1 or STAT5A(1*6) as described in Fig. 1 and grown in cocultures on stromal cell lines as indicated. (A) Representative examples of CAFCs at day 10 observed in coculture studies with CB CD34 + cells transduced with STAT5A(1*6). (B) CAFC frequencies were determined on MS5 in limiting dilution conditions as described in Materials and Methods. (C) GFP + cells were sorted from the adherent population on MS5 at day 10 and used for FACS analysis and cytospins (D). (E) CB CD34 + cells were transduced with MiGR1 or STAT5A(1*6), after which the CD38 high and CD38 low populations were sorted and used to determine the CAFC frequencies on MS5 under limiting dilution conditions. First CAFC frequencies were determined at day 10, and cultures were either harvested and plated on new MS5 stroma to determine secondary CAFC frequencies (indicated in black as number of secondary CAFCs/number of primary CAFCs), or plated in methylcellulose to determine the number of CFCs/10 CAFCs (F). (G) CB CD34 + cells were transduced with MiGR1 or STAT5A(1*6) as described in Fig. 1 and plated for CFC assays in triplicate. After 2 wk, the colonies were scored and cells from each plate were plated for secondary CFCs. (H) Sublethally irradiated NOD-SCID recipients were injected with 2 × 10 5 GFP + day 10 STAT5A(1*6) CAFCs. Mice were killed at week 6 and the bone marrow was analyzed as indicated.

    Techniques Used: Transduction, FACS, Irradiation, Injection, Mouse Assay

    20) Product Images from "Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum"

    Article Title: Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016619

    Time course for the in vitro induction of native Nr4a1 mRNA and protein compared to Nr4a1 promoter-driven eGFP expression in MSNs. Semi-quantitative PCR detection of Nr4a1, eGFP and actin mRNA after exposure to 30 mM KCl for 30 min to 8 hrs is shown in A. Western blot detection of eGFP, Nr4a1 and actin induced by 30 mM KCl or forskolin (1 hr to 8 hr) is shown in panel B. Immunofluorescent detection Nr4a1 (red) and eGFP in DAPI stained MSN cultures is shown in C1–C4 after 2 hrs of treatment with 30 mM KCl (C1, C2) or 8 hrs (C3, C4). Arrows in C1 indicate nuclear expression in brightly fluorescent cells after 2 hrs of exposure to 30 mM KCl. DAPI and Nr4a1 are shown together in C2 for this time point. Cells treated for 8 hrs with 30 mM KCl are shown in C3,C4. Arrows (C3) indicate cells with perinuclear eGFP and Nr4a1 immunoreactivity. The DAPI and Nr4a1 channels are shown merged in C4. Arrows (C4) indicate the absence of Nr4a1 immunoreactivity in the nuclei of brightly eGFP-expressing cells at 8 hrs. Scale bar in C1 (10 µm) applies to all images.
    Figure Legend Snippet: Time course for the in vitro induction of native Nr4a1 mRNA and protein compared to Nr4a1 promoter-driven eGFP expression in MSNs. Semi-quantitative PCR detection of Nr4a1, eGFP and actin mRNA after exposure to 30 mM KCl for 30 min to 8 hrs is shown in A. Western blot detection of eGFP, Nr4a1 and actin induced by 30 mM KCl or forskolin (1 hr to 8 hr) is shown in panel B. Immunofluorescent detection Nr4a1 (red) and eGFP in DAPI stained MSN cultures is shown in C1–C4 after 2 hrs of treatment with 30 mM KCl (C1, C2) or 8 hrs (C3, C4). Arrows in C1 indicate nuclear expression in brightly fluorescent cells after 2 hrs of exposure to 30 mM KCl. DAPI and Nr4a1 are shown together in C2 for this time point. Cells treated for 8 hrs with 30 mM KCl are shown in C3,C4. Arrows (C3) indicate cells with perinuclear eGFP and Nr4a1 immunoreactivity. The DAPI and Nr4a1 channels are shown merged in C4. Arrows (C4) indicate the absence of Nr4a1 immunoreactivity in the nuclei of brightly eGFP-expressing cells at 8 hrs. Scale bar in C1 (10 µm) applies to all images.

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

    Native fluorescence and fluorometric detection of stimulated eGFP expression in primary cultures of medium spiny neurons stimulated in vitro. Pseudocolored images of control (A), 5 ng/mL BDNF (B), 1 µM forskolin (C), 1 µM SKF-83822 (D) or 30 mM KCl (E) reveal eGFP induction after 20 hrs. eGFP expression was also measured fluorometrically in lysates at 3, 8 and 20 hrs after exposure (F). Native fluorescence is representative of 4 replicate fields taken at the same exposure time (set for linear detection in control cultures). Fluorometry data in F are the mean +/− SE of 16 replicates for each time point and treatment. Data were analyzed by ANOVA (* p
    Figure Legend Snippet: Native fluorescence and fluorometric detection of stimulated eGFP expression in primary cultures of medium spiny neurons stimulated in vitro. Pseudocolored images of control (A), 5 ng/mL BDNF (B), 1 µM forskolin (C), 1 µM SKF-83822 (D) or 30 mM KCl (E) reveal eGFP induction after 20 hrs. eGFP expression was also measured fluorometrically in lysates at 3, 8 and 20 hrs after exposure (F). Native fluorescence is representative of 4 replicate fields taken at the same exposure time (set for linear detection in control cultures). Fluorometry data in F are the mean +/− SE of 16 replicates for each time point and treatment. Data were analyzed by ANOVA (* p

    Techniques Used: Fluorescence, Expressing, In Vitro

    21) Product Images from "CA8 Mutations Cause a Novel Syndrome Characterized by Ataxia and Mild Mental Retardation with Predisposition to Quadrupedal Gait"

    Article Title: CA8 Mutations Cause a Novel Syndrome Characterized by Ataxia and Mild Mental Retardation with Predisposition to Quadrupedal Gait

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000487

    The mutation S100P reduces CA8 protein stability by means of proteasome-mediated CA8 degradation. (A) Reduction of CA8 protein concentration by S100P. Production of wildtype and mutant CA8 was induced by tetracycline. There was a strong reduction in the level of mutant CA8 protein compared to that of the wildtype at both 0.1, and 1.0 µg/ml tetracycline. *: , **: , comparison between mutant and wildtype at the indicated tetracycline concentration by two-sided t -test. The expression is represented as signal intensity ratio between CA8/GAPDH, which was normalized to the WT level induced by 1 µg/ml tetracycline. Mean values±standard deviation (SD) in triplicate experiments are shown. (B) Rescue of mutant CA8 protein expression by proteasomal inhibition. Addition of the proteasome inhibitor MG132 lead to a dose-dependent rescue of CA8 concentration. *: , comparison between 0 µM MG132 and 0.4 µM or 2.0 µM MG132. Expression is shown as ratio relative to that of the mutant CA8 protein (S100P) without MG132. Mean values±SD in triplicate experiments are shown.
    Figure Legend Snippet: The mutation S100P reduces CA8 protein stability by means of proteasome-mediated CA8 degradation. (A) Reduction of CA8 protein concentration by S100P. Production of wildtype and mutant CA8 was induced by tetracycline. There was a strong reduction in the level of mutant CA8 protein compared to that of the wildtype at both 0.1, and 1.0 µg/ml tetracycline. *: , **: , comparison between mutant and wildtype at the indicated tetracycline concentration by two-sided t -test. The expression is represented as signal intensity ratio between CA8/GAPDH, which was normalized to the WT level induced by 1 µg/ml tetracycline. Mean values±standard deviation (SD) in triplicate experiments are shown. (B) Rescue of mutant CA8 protein expression by proteasomal inhibition. Addition of the proteasome inhibitor MG132 lead to a dose-dependent rescue of CA8 concentration. *: , comparison between 0 µM MG132 and 0.4 µM or 2.0 µM MG132. Expression is shown as ratio relative to that of the mutant CA8 protein (S100P) without MG132. Mean values±SD in triplicate experiments are shown.

    Techniques Used: Mutagenesis, Protein Concentration, Concentration Assay, Expressing, Standard Deviation, Inhibition

    22) Product Images from "Restraint Stress Intensifies Interstitial K+ Accumulation during Severe Hypoxia"

    Article Title: Restraint Stress Intensifies Interstitial K+ Accumulation during Severe Hypoxia

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2012.00053

    Quantification of GFAP and Kir4.1 expression . (A) Immunolabeling of the astrocytic marker GFAP revealed an increased GFAP immunoreactivity in the hippocampal CA1 subfield in sections from stressed rats. Relative optical density of the sections was determined in st. oriens and st. radiatum (bar plots on the right). In both layers, GFAP immunoreactivity was more dense in stressed as compared to control rats. The number of sections analyzed is reported (so st. oriens , sp st. pyramidale , sr st. radiatum) . (B) Immunolabeling also revealed a downregulation of Kir4.1 in stressed rats that was obvious in all layers of the CA1 subfield. (C) Western blots confirmed the decreased Kir4.1 immunoreactivity, yielding a decreased expression of Kir4.1 as compared to β-actin content in stressed rats ( n = 8 hippocampi each group).
    Figure Legend Snippet: Quantification of GFAP and Kir4.1 expression . (A) Immunolabeling of the astrocytic marker GFAP revealed an increased GFAP immunoreactivity in the hippocampal CA1 subfield in sections from stressed rats. Relative optical density of the sections was determined in st. oriens and st. radiatum (bar plots on the right). In both layers, GFAP immunoreactivity was more dense in stressed as compared to control rats. The number of sections analyzed is reported (so st. oriens , sp st. pyramidale , sr st. radiatum) . (B) Immunolabeling also revealed a downregulation of Kir4.1 in stressed rats that was obvious in all layers of the CA1 subfield. (C) Western blots confirmed the decreased Kir4.1 immunoreactivity, yielding a decreased expression of Kir4.1 as compared to β-actin content in stressed rats ( n = 8 hippocampi each group).

    Techniques Used: Expressing, Immunolabeling, Marker, Western Blot

    23) Product Images from "Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis"

    Article Title: Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv259

    Examination of DNA binding mutants of Hop2-Mnd1 in the D-loop reaction catalyzed by RAD51. (a) Schematic of the D-loop reaction ( 23 ). (b) Wild type (WT) and mutant variants of the Hop2-Mnd1 complex (60 nM, 120 nM, 180 nM or 240 nM) were tested for their ability to promote D-loop formation with ATP as nucleotide cofactor. (c) WT and mutant variants of the Hop2-Mnd1 complex (60 nM or 180 nM) were tested for their ability to promote D-loop formation under three different sets of conditions: Ca 2+ +Mg 2+ +ATP (left panel), Mg 2+ +ATP (middle panel) and Mg 2+ +AMP-PNP (right panel). In (b) and (c), the mean values ± s.d. from three independent experiments were plotted.
    Figure Legend Snippet: Examination of DNA binding mutants of Hop2-Mnd1 in the D-loop reaction catalyzed by RAD51. (a) Schematic of the D-loop reaction ( 23 ). (b) Wild type (WT) and mutant variants of the Hop2-Mnd1 complex (60 nM, 120 nM, 180 nM or 240 nM) were tested for their ability to promote D-loop formation with ATP as nucleotide cofactor. (c) WT and mutant variants of the Hop2-Mnd1 complex (60 nM or 180 nM) were tested for their ability to promote D-loop formation under three different sets of conditions: Ca 2+ +Mg 2+ +ATP (left panel), Mg 2+ +ATP (middle panel) and Mg 2+ +AMP-PNP (right panel). In (b) and (c), the mean values ± s.d. from three independent experiments were plotted.

    Techniques Used: Binding Assay, Mutagenesis

    The robust interaction of Hop2-Mnd1 with RAD51 is induced by ATP and requires both subunits of the Hop2-Mnd1 complex. (a) Affinity pulldown to test for the interaction between RAD51 and (His) 6 -tagged Hop2-Mnd1 or (His) 6 -tagged Mnd1 using Ni 2+ -NTA resin to capture protein complexes. The reactions contained the indicated concentration of KCl and with or without ATP. The supernatant (S) containing unbound proteins, the wash (W) and the eluate (E) fractions were analyzed by SDS-PAGE and Coomassie Blue staining. (b) Yeast three-hybrid analysis to test for the interaction of RAD51 with Hop2, Mnd1 and Hop2-Mnd1. Abbreviations: H2, Hop2; M1, Mnd1; 51, RAD51.
    Figure Legend Snippet: The robust interaction of Hop2-Mnd1 with RAD51 is induced by ATP and requires both subunits of the Hop2-Mnd1 complex. (a) Affinity pulldown to test for the interaction between RAD51 and (His) 6 -tagged Hop2-Mnd1 or (His) 6 -tagged Mnd1 using Ni 2+ -NTA resin to capture protein complexes. The reactions contained the indicated concentration of KCl and with or without ATP. The supernatant (S) containing unbound proteins, the wash (W) and the eluate (E) fractions were analyzed by SDS-PAGE and Coomassie Blue staining. (b) Yeast three-hybrid analysis to test for the interaction of RAD51 with Hop2, Mnd1 and Hop2-Mnd1. Abbreviations: H2, Hop2; M1, Mnd1; 51, RAD51.

    Techniques Used: Concentration Assay, SDS Page, Staining

    Hop2 and Mnd1 mutants defective in interaction with RAD51. (a) Alignment of the C-termini of Mnd1 orthologs. The arrowheads highlight the residues selected as mutagenesis targets. (b) Alignment of the C-termini of Hop2 orthologs. The arrowheads highlight the residues picked as mutagenesis targets (red arrowheads denote those residues that impair recombinase interaction when mutated; white arrowheads denote those mutations that do not affect recombinase interaction when mutated). The E201 residue that is deleted in a case of XX-GD is denoted by the asterisk. Note that there is only limited overall sequence similarity between the yeast and mammalian Hop2 and Mnd1 species in their C-terminal region (a and b), which could explain why the yeast Hop2–Mnd1 complex does not interact with Rad51. (c) Pulldown assay to test for the interaction of RAD51 with MBP-tagged Hop2-Mnd1 and mutants using amylose resin to capture protein complexes. Analysis was conducted as in Figure 4a . (d) Wild type (WT) and mutant variants of the Hop2-Mnd1 complex (90 or 180 nM) were tested for their ability to promote D-loop formation by RAD51. The mean values ± s.d. from three independent experiments were plotted. Abbreviations in (a) and (b): Mm, Mus musculus ; Hs, Homo sapiens ; At, Arabidopsis thaliana ; Sp, Schizosaccharomyces pombe ; Sc, Saccharomyces cerevisiae .
    Figure Legend Snippet: Hop2 and Mnd1 mutants defective in interaction with RAD51. (a) Alignment of the C-termini of Mnd1 orthologs. The arrowheads highlight the residues selected as mutagenesis targets. (b) Alignment of the C-termini of Hop2 orthologs. The arrowheads highlight the residues picked as mutagenesis targets (red arrowheads denote those residues that impair recombinase interaction when mutated; white arrowheads denote those mutations that do not affect recombinase interaction when mutated). The E201 residue that is deleted in a case of XX-GD is denoted by the asterisk. Note that there is only limited overall sequence similarity between the yeast and mammalian Hop2 and Mnd1 species in their C-terminal region (a and b), which could explain why the yeast Hop2–Mnd1 complex does not interact with Rad51. (c) Pulldown assay to test for the interaction of RAD51 with MBP-tagged Hop2-Mnd1 and mutants using amylose resin to capture protein complexes. Analysis was conducted as in Figure 4a . (d) Wild type (WT) and mutant variants of the Hop2-Mnd1 complex (90 or 180 nM) were tested for their ability to promote D-loop formation by RAD51. The mean values ± s.d. from three independent experiments were plotted. Abbreviations in (a) and (b): Mm, Mus musculus ; Hs, Homo sapiens ; At, Arabidopsis thaliana ; Sp, Schizosaccharomyces pombe ; Sc, Saccharomyces cerevisiae .

    Techniques Used: Mutagenesis, Sequencing

    Role of the Hop2 C-terminal DNA binding domain in RAD51 presynaptic filament stabilization. (a) Schematic of the presynaptic filament stabilization assay ( 13 ). (b) Wild type (WT) and mutant variants of the Hop2–Mnd1 complex were tested for their ability to stabilize the RAD51 presynaptic filament. The mean values ± s.d. from three independent experiments were plotted.
    Figure Legend Snippet: Role of the Hop2 C-terminal DNA binding domain in RAD51 presynaptic filament stabilization. (a) Schematic of the presynaptic filament stabilization assay ( 13 ). (b) Wild type (WT) and mutant variants of the Hop2–Mnd1 complex were tested for their ability to stabilize the RAD51 presynaptic filament. The mean values ± s.d. from three independent experiments were plotted.

    Techniques Used: Binding Assay, Mutagenesis

    Characterization of Hop2-Mnd1 with the XX-GD HOP2 p.Glu201del (ΔE201) mutation. (a) Pulldown assay to test for the interaction of RAD51 (left panel) or DMC1 (right panel) with MBP-tagged Hop2-Mnd1 and Hop2-ΔE201-Mnd1 using amylose resin to capture protein complexes. Analysis was conducted as in Figure 4a . (b) Hop2-Mnd1 and Hop2-ΔE201-Mnd1 (90 and 180 nM for RAD51; 60, 90 and 120 nM for DMC1) were tested for their ability to promote D-loop formation by RAD51 (left panel) or DMC1 (right panel). The mean values ± s.d. from three independent experiments were plotted.
    Figure Legend Snippet: Characterization of Hop2-Mnd1 with the XX-GD HOP2 p.Glu201del (ΔE201) mutation. (a) Pulldown assay to test for the interaction of RAD51 (left panel) or DMC1 (right panel) with MBP-tagged Hop2-Mnd1 and Hop2-ΔE201-Mnd1 using amylose resin to capture protein complexes. Analysis was conducted as in Figure 4a . (b) Hop2-Mnd1 and Hop2-ΔE201-Mnd1 (90 and 180 nM for RAD51; 60, 90 and 120 nM for DMC1) were tested for their ability to promote D-loop formation by RAD51 (left panel) or DMC1 (right panel). The mean values ± s.d. from three independent experiments were plotted.

    Techniques Used: Mutagenesis

    Model for the mechanism of the Hop2-Mnd1-RAD51/DMC1-ssDNA ensemble. (a) Cartoon showing the Hop2-Mnd1 complex and functional domains in the protein subunits. (b) Our model posits that Hop2–Mnd1 associates with RAD51/DMC1 through an interface contributed by the C-termini of Hop2 and Mnd1, stabilizes the RAD51/DMC1 presynaptic filament via its ssDNA-binding activity within the C-terminus of Hop2 and helps capture duplex DNA by its dsDNA binding functions located in the N-termini of Hop2 and Mnd1. These attributes of Hop2-Mnd1 enhance the efficiency at which a duplex DNA molecule is engaged and sampled for homology by the RAD51/DMC1 presynaptic filament.
    Figure Legend Snippet: Model for the mechanism of the Hop2-Mnd1-RAD51/DMC1-ssDNA ensemble. (a) Cartoon showing the Hop2-Mnd1 complex and functional domains in the protein subunits. (b) Our model posits that Hop2–Mnd1 associates with RAD51/DMC1 through an interface contributed by the C-termini of Hop2 and Mnd1, stabilizes the RAD51/DMC1 presynaptic filament via its ssDNA-binding activity within the C-terminus of Hop2 and helps capture duplex DNA by its dsDNA binding functions located in the N-termini of Hop2 and Mnd1. These attributes of Hop2-Mnd1 enhance the efficiency at which a duplex DNA molecule is engaged and sampled for homology by the RAD51/DMC1 presynaptic filament.

    Techniques Used: Functional Assay, Binding Assay, Activity Assay

    24) Product Images from "Elf-1 Contributes to the Function of the Complex Interleukin (IL)-2-responsive Enhancer in the Mouse IL-2 Receptor ? Gene"

    Article Title: Elf-1 Contributes to the Function of the Complex Interleukin (IL)-2-responsive Enhancer in the Mouse IL-2 Receptor ? Gene

    Journal: The Journal of Experimental Medicine

    doi:

    Elf-1 is precipitated from PC60–derived nuclear extracts by biotinylated IL-2rE oligonucleotide probes containing an intact site III. The ability of various biotinylated IL-2rE oligonucleotide probes to precipitate Elf-1 from PC60-derived nuclear extracts was investigated. The nature of the IL-2rE probe used to precipitate the protein is indicated below the lane(s); a blank or crossed box, respectively, serves to illustrate the presence of either a wild-type or a mutant sequence at the designated site of the IL-2rE enhancer (the mutant forms of sites I, II, and III contain the sequences M4, M9, and M12, respectively, which destroy IL-2rE enhancer activity, see Fig. 1 A and Table 1 ). In A and C nuclear extracts were prepared from cells that had been IL-1 primed and then IL-2 induced for the specified time periods. In B , all extracts were prepared from cells that had been IL-1 primed and IL-2 induced for 30 min. The blots were probed with anti–Elf-1 antibodies and molecular mass markers are indicated.
    Figure Legend Snippet: Elf-1 is precipitated from PC60–derived nuclear extracts by biotinylated IL-2rE oligonucleotide probes containing an intact site III. The ability of various biotinylated IL-2rE oligonucleotide probes to precipitate Elf-1 from PC60-derived nuclear extracts was investigated. The nature of the IL-2rE probe used to precipitate the protein is indicated below the lane(s); a blank or crossed box, respectively, serves to illustrate the presence of either a wild-type or a mutant sequence at the designated site of the IL-2rE enhancer (the mutant forms of sites I, II, and III contain the sequences M4, M9, and M12, respectively, which destroy IL-2rE enhancer activity, see Fig. 1 A and Table 1 ). In A and C nuclear extracts were prepared from cells that had been IL-1 primed and then IL-2 induced for the specified time periods. In B , all extracts were prepared from cells that had been IL-1 primed and IL-2 induced for 30 min. The blots were probed with anti–Elf-1 antibodies and molecular mass markers are indicated.

    Techniques Used: Derivative Assay, Mutagenesis, Sequencing, Activity Assay

    25) Product Images from "Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements"

    Article Title: Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl543

    Distributions of hyperacetylated H2A.Z at the lysozyme locus in HD37 erythroblasts and untreated HD11 macrophages. The distribution, more precisely the absence, of unacetylated H2A.Z is shown at the bottom of the figure. Nomenclature as in Figure 3 .
    Figure Legend Snippet: Distributions of hyperacetylated H2A.Z at the lysozyme locus in HD37 erythroblasts and untreated HD11 macrophages. The distribution, more precisely the absence, of unacetylated H2A.Z is shown at the bottom of the figure. Nomenclature as in Figure 3 .

    Techniques Used:

    26) Product Images from "Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements"

    Article Title: Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl543

    Proteins from an nChIP using mononucleosomes from HD11 cells and anti-hyperacetylated H4 antibodies, analysed on an acetic acid/urea/Triton (AUT) gel. B , bound fraction; U , unbound fraction; I , input fraction. Note that in addition to the expected enrichment of hyperacetylated species of H4, the Ab-bound nucleosomes are also enriched in acetylated species of H2B and also the replacement histone H2A.Z.
    Figure Legend Snippet: Proteins from an nChIP using mononucleosomes from HD11 cells and anti-hyperacetylated H4 antibodies, analysed on an acetic acid/urea/Triton (AUT) gel. B , bound fraction; U , unbound fraction; I , input fraction. Note that in addition to the expected enrichment of hyperacetylated species of H4, the Ab-bound nucleosomes are also enriched in acetylated species of H2B and also the replacement histone H2A.Z.

    Techniques Used:

    Distributions of hyperacetylated H2A.Z at the lysozyme locus in HD37 erythroblasts and untreated HD11 macrophages. The distribution, more precisely the absence, of unacetylated H2A.Z is shown at the bottom of the figure. Nomenclature as in Figure 3 .
    Figure Legend Snippet: Distributions of hyperacetylated H2A.Z at the lysozyme locus in HD37 erythroblasts and untreated HD11 macrophages. The distribution, more precisely the absence, of unacetylated H2A.Z is shown at the bottom of the figure. Nomenclature as in Figure 3 .

    Techniques Used:

    27) Product Images from "Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering"

    Article Title: Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering

    Journal: Genome Integrity

    doi: 10.1186/2041-9414-1-6

    Construction of truncated chromosome 3 in DT40 cells by telomere seeding . (A) Targeting strategy of generating truncated chromosome. (B) Summary of PCR analyses on truncated chromosomes in DT40 cells. Twenty STS markers on chromosome 3 examined were shown. Solid circles and open circles represent presence and absence of truncated allele at tested loci in DT40 cells, respectively.
    Figure Legend Snippet: Construction of truncated chromosome 3 in DT40 cells by telomere seeding . (A) Targeting strategy of generating truncated chromosome. (B) Summary of PCR analyses on truncated chromosomes in DT40 cells. Twenty STS markers on chromosome 3 examined were shown. Solid circles and open circles represent presence and absence of truncated allele at tested loci in DT40 cells, respectively.

    Techniques Used: Polymerase Chain Reaction

    FISH analysis of truncated human chromosome 3 in DT40 cells . Upper panels show representative metaphase spreads (A-D) and bottom panels show the enlarge image of human chromosome with allow. The allow shows a targeting site on chromosome 3 using biotin-labeled PGK-puro probe ( green color ). (A) DT40 containing an intact human chromosome 3. (B) DT40 containing #3delp24-pter. (C) DT40 containing #3delp22-pter. (D) DT40 containing #3delp21.3-pter. Digoxigenin-labeled human COT-1 probe ( red color ) indicates human chromosome. DAPI staining ( blue color ) shows chicken chromosomes.
    Figure Legend Snippet: FISH analysis of truncated human chromosome 3 in DT40 cells . Upper panels show representative metaphase spreads (A-D) and bottom panels show the enlarge image of human chromosome with allow. The allow shows a targeting site on chromosome 3 using biotin-labeled PGK-puro probe ( green color ). (A) DT40 containing an intact human chromosome 3. (B) DT40 containing #3delp24-pter. (C) DT40 containing #3delp22-pter. (D) DT40 containing #3delp21.3-pter. Digoxigenin-labeled human COT-1 probe ( red color ) indicates human chromosome. DAPI staining ( blue color ) shows chicken chromosomes.

    Techniques Used: Fluorescence In Situ Hybridization, Labeling, Staining

    28) Product Images from "Pharmacologically modified pluripotent stem cell-based cancer vaccines with anti-metastatic potential"

    Article Title: Pharmacologically modified pluripotent stem cell-based cancer vaccines with anti-metastatic potential

    Journal: bioRxiv

    doi: 10.1101/2020.05.27.118471

    Vaccination with embryonic stem cells elicits anti-tumoral effect in vivo (A) Vaccination and challenge protocol: irradiated stem cells or 4T1 cells were injected as a vaccine into BALB/c mice twice, with a one-week interval between doses. Mice were then challenged with 5×10 4 4T1 cells one week after the final dose. (B-D) hESC-, mESC-, and 4T1-vaccinated groups had significantly (p
    Figure Legend Snippet: Vaccination with embryonic stem cells elicits anti-tumoral effect in vivo (A) Vaccination and challenge protocol: irradiated stem cells or 4T1 cells were injected as a vaccine into BALB/c mice twice, with a one-week interval between doses. Mice were then challenged with 5×10 4 4T1 cells one week after the final dose. (B-D) hESC-, mESC-, and 4T1-vaccinated groups had significantly (p

    Techniques Used: In Vivo, Irradiation, Injection, Mouse Assay

    Effective memory immune response following vaccination with miPSCs (A) Experimental protocol to evaluate in vivo immune memory generated by vaccination: BALB/c mice were injected subcutaneously six times with 2×10 6 miPSCs (15Gy irradiated) in the right flank. Tumor challenge occurred 120 days after the final vaccine dose was given; mice were injected with 2.5×10 4 4T1-GFP-Luc cells and VPA was added in their drinking water. (B) At day 28 post-challenge, breast tumors were significantly smaller in mice that had undergone the six-month vaccination protocol compared to unvaccinated mice (n=6 per group). (C) Bioluminescence images of lungs isolated from miPSC-vaccinated and control mice 28 days after tumor challenge. (D) A significant correlation was found between tumor burden and metastatic spread in the lungs of vaccinated mice at day 28 post-challenge. (E) The frequency of CD4 + CD8 + cells in tumors of treated and untreated mice, as measured by flow cytometry. (F) The frequency of PDL1 + cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (G) The frequency of Treg cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (H) The frequency of Arg1 + preMDSCs and granMDSCs in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (I) The frequency of CXCR5 + CD22.2 + LB cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (J) The frequency of T-effector memory cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (K) The frequency of KLRG1 + T-effector memory cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry.
    Figure Legend Snippet: Effective memory immune response following vaccination with miPSCs (A) Experimental protocol to evaluate in vivo immune memory generated by vaccination: BALB/c mice were injected subcutaneously six times with 2×10 6 miPSCs (15Gy irradiated) in the right flank. Tumor challenge occurred 120 days after the final vaccine dose was given; mice were injected with 2.5×10 4 4T1-GFP-Luc cells and VPA was added in their drinking water. (B) At day 28 post-challenge, breast tumors were significantly smaller in mice that had undergone the six-month vaccination protocol compared to unvaccinated mice (n=6 per group). (C) Bioluminescence images of lungs isolated from miPSC-vaccinated and control mice 28 days after tumor challenge. (D) A significant correlation was found between tumor burden and metastatic spread in the lungs of vaccinated mice at day 28 post-challenge. (E) The frequency of CD4 + CD8 + cells in tumors of treated and untreated mice, as measured by flow cytometry. (F) The frequency of PDL1 + cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (G) The frequency of Treg cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (H) The frequency of Arg1 + preMDSCs and granMDSCs in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (I) The frequency of CXCR5 + CD22.2 + LB cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (J) The frequency of T-effector memory cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry. (K) The frequency of KLRG1 + T-effector memory cells in tumors of miPSC-vaccinated mice compared to controls, as measured by flow cytometry.

    Techniques Used: In Vivo, Generated, Mouse Assay, Injection, Irradiation, Isolation, Flow Cytometry

    Breast tumors induced by 4T1 cells demonstrate an ESC-like expression signature in vitro and in vivo (A) Transcriptome heatmap showing genes that were overexpressed in implanted 4T1 cells and in D3 murine embryonic stem cells (Euclidean distances, complete method). (B) Functional enrichment analysis of 85 genes that were overexpressed in implanted 4T1 cells and in D3 murine embryonic stem cells (Gene Ontology database); bars represent negative log10 of enrichment p-values. (C) Quantification of CD44/CD24 markers in 4T1 cells by flow cytometry in vitro and in vivo 12 and 28 days after implantation into the fad pat of BALB/c mice.
    Figure Legend Snippet: Breast tumors induced by 4T1 cells demonstrate an ESC-like expression signature in vitro and in vivo (A) Transcriptome heatmap showing genes that were overexpressed in implanted 4T1 cells and in D3 murine embryonic stem cells (Euclidean distances, complete method). (B) Functional enrichment analysis of 85 genes that were overexpressed in implanted 4T1 cells and in D3 murine embryonic stem cells (Gene Ontology database); bars represent negative log10 of enrichment p-values. (C) Quantification of CD44/CD24 markers in 4T1 cells by flow cytometry in vitro and in vivo 12 and 28 days after implantation into the fad pat of BALB/c mice.

    Techniques Used: Expressing, In Vitro, In Vivo, Functional Assay, Flow Cytometry, Mouse Assay

    Evaluation of the anti-tumor effects of murine iPSCs derived from BALB/c or C57Bl/6 mice. (A) Tumor growth in mice that were immunized with murine BALB/c-derived iPSCs, with or without VPA, compared to control group. (B) Tumor growth in mice that were immunized with murine C57BL/6-derived iPSCs, with or without VPA, compared to control group. (C) Effect of allogeneic miPSC treatment on the survival of mice challenged with 4T1 cells. (D) Effect of autologous miPSC treatment on the survival of mice challenged with 4T1 cells. (E) Effect of allogeneic miPSC+VPA treatment on the survival of mice challenged with 4T1 cells. (F) Effect of autologous miPSC+VPA treatment on the survival of mice challenged with 4T1 cells. (G) Tumor volumes of mice treated with allogeneic miPSCs+VPA compared to those of untreated mice. The data represent the mean + SEM of tumor volumes (8 mice per group).
    Figure Legend Snippet: Evaluation of the anti-tumor effects of murine iPSCs derived from BALB/c or C57Bl/6 mice. (A) Tumor growth in mice that were immunized with murine BALB/c-derived iPSCs, with or without VPA, compared to control group. (B) Tumor growth in mice that were immunized with murine C57BL/6-derived iPSCs, with or without VPA, compared to control group. (C) Effect of allogeneic miPSC treatment on the survival of mice challenged with 4T1 cells. (D) Effect of autologous miPSC treatment on the survival of mice challenged with 4T1 cells. (E) Effect of allogeneic miPSC+VPA treatment on the survival of mice challenged with 4T1 cells. (F) Effect of autologous miPSC+VPA treatment on the survival of mice challenged with 4T1 cells. (G) Tumor volumes of mice treated with allogeneic miPSCs+VPA compared to those of untreated mice. The data represent the mean + SEM of tumor volumes (8 mice per group).

    Techniques Used: Derivative Assay, Mouse Assay

    29) Product Images from "Phosphorylation by Aurora B kinase regulates caspase-2 activity and function"

    Article Title: Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

    Journal: bioRxiv

    doi: 10.1101/2020.03.05.978056

    Caspase-2-S384E mutant does not cleave MDM2 or Bid. a. U2OS- CASP2 -/- cells were transfected with GFP mock vector, GFP-caspase-2 WT, C320G or phosphorylation site mutants. Cell lysates were subjected to immunoblotting. with the indicated antibodies. β-actin was used as loading control. b. U2OS- CASP2 -/- cells were co-transfected with GFP mock vector, GFP-caspase-2 WT, C320G or phosphorylation site mutants and with Bid-HA. Cell lysates were subjected to immunoblotting with the indicated antibodies. β-actin was used as loading control . c and d. For cell death assays, Casp2 -/- immortalised MEFs were co-transfected with GFP-caspase-2 WT, C320G, S384A or S384E and β-gal expressing plasmids. After 24 h, cells were fixed and incubated with an X-gal containing solution. Blue (transfected) cells were counted for apoptotic morphology under microscope. c. Rrepresentative images showing X-gal staining with live or apoptotic cell morphology. Arrow, dead cell; arrowhead, live cell. Scale bar=50μm d . Graph showing % cell death. *, vs. WT; ****, p
    Figure Legend Snippet: Caspase-2-S384E mutant does not cleave MDM2 or Bid. a. U2OS- CASP2 -/- cells were transfected with GFP mock vector, GFP-caspase-2 WT, C320G or phosphorylation site mutants. Cell lysates were subjected to immunoblotting. with the indicated antibodies. β-actin was used as loading control. b. U2OS- CASP2 -/- cells were co-transfected with GFP mock vector, GFP-caspase-2 WT, C320G or phosphorylation site mutants and with Bid-HA. Cell lysates were subjected to immunoblotting with the indicated antibodies. β-actin was used as loading control . c and d. For cell death assays, Casp2 -/- immortalised MEFs were co-transfected with GFP-caspase-2 WT, C320G, S384A or S384E and β-gal expressing plasmids. After 24 h, cells were fixed and incubated with an X-gal containing solution. Blue (transfected) cells were counted for apoptotic morphology under microscope. c. Rrepresentative images showing X-gal staining with live or apoptotic cell morphology. Arrow, dead cell; arrowhead, live cell. Scale bar=50μm d . Graph showing % cell death. *, vs. WT; ****, p

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Incubation, Microscopy, Staining

    Caspase-2-S384E expressing cells are resistant to aberrant mitosis-mediated cell death. GFP-expressing U2OS (sWT) and U2OS- CASP2 -/- expressing GFP (sKO), GFP-caspase-2-C320G (sC320G) or GFP-caspase-2-S384E (sS384 #1 and #2), were treated with DMSO or 100 nM BI 2536 (BI) for 48 h and subjected to immunoblot and MTS assay. a. Plot showing percentage of viable cells following BI treatment. mean ± SEM; n=3. *, vs. WT; *, p
    Figure Legend Snippet: Caspase-2-S384E expressing cells are resistant to aberrant mitosis-mediated cell death. GFP-expressing U2OS (sWT) and U2OS- CASP2 -/- expressing GFP (sKO), GFP-caspase-2-C320G (sC320G) or GFP-caspase-2-S384E (sS384 #1 and #2), were treated with DMSO or 100 nM BI 2536 (BI) for 48 h and subjected to immunoblot and MTS assay. a. Plot showing percentage of viable cells following BI treatment. mean ± SEM; n=3. *, vs. WT; *, p

    Techniques Used: Expressing, MTS Assay

    Caspase-2-S384E expressing cells fail to cleave MDM2 and show increased polyploidy following AURBK inhibition. GFP-expressing U2OS (sWT) and U2OS- CASP2 -/- expressing GFP (sKO), GFP-caspase-2-C320G (sC320G) or GFP-caspase-2-S384E (sS384 #1 and #2) were treated with DMSO or 2 µM ZM447439 (ZM) for 48 h and subjected to immunoblot and DNA content analysis. a. Representative immunoblots (of three independent experiments performed) of cell lysates from treated stable cell lines. Antibodies used for immunoblotting are as indicated. β-actin was used as loading control. b. Representative flow cytometric profiles of the DNA content in cells following ZM treatment. Percentage of polyploid cells ( > 4n) is indicated. c. Graph comparing percentage of cells with polyploid ( > 4N) DNA content following ZM treatment. mean ± SEM; n=3. *, vs. WT + ZM; *, p
    Figure Legend Snippet: Caspase-2-S384E expressing cells fail to cleave MDM2 and show increased polyploidy following AURBK inhibition. GFP-expressing U2OS (sWT) and U2OS- CASP2 -/- expressing GFP (sKO), GFP-caspase-2-C320G (sC320G) or GFP-caspase-2-S384E (sS384 #1 and #2) were treated with DMSO or 2 µM ZM447439 (ZM) for 48 h and subjected to immunoblot and DNA content analysis. a. Representative immunoblots (of three independent experiments performed) of cell lysates from treated stable cell lines. Antibodies used for immunoblotting are as indicated. β-actin was used as loading control. b. Representative flow cytometric profiles of the DNA content in cells following ZM treatment. Percentage of polyploid cells ( > 4n) is indicated. c. Graph comparing percentage of cells with polyploid ( > 4N) DNA content following ZM treatment. mean ± SEM; n=3. *, vs. WT + ZM; *, p

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

    Caspase-2-S384E mutant can still homo-dimerise but lacks enzymatic activity. a. Representative confocal images showing dimerisation of Casp2-S384E or CASP2-C320A by BiFC. U2OS- CASP2 -/- cells were transfected with Casp2-S384E or CASP2-C320A BiFC constructs, respectively, and treated with DMSO or 100 nM BI2536 (BI) for 24h. All cells were also treated with zVAD (20 µM), pan-caspase inhibitor. Arrows indicate dimerised caspase-2. Scale bar=20μm. b. Quantitation of relative BiFC positive cells are indicated. *, P
    Figure Legend Snippet: Caspase-2-S384E mutant can still homo-dimerise but lacks enzymatic activity. a. Representative confocal images showing dimerisation of Casp2-S384E or CASP2-C320A by BiFC. U2OS- CASP2 -/- cells were transfected with Casp2-S384E or CASP2-C320A BiFC constructs, respectively, and treated with DMSO or 100 nM BI2536 (BI) for 24h. All cells were also treated with zVAD (20 µM), pan-caspase inhibitor. Arrows indicate dimerised caspase-2. Scale bar=20μm. b. Quantitation of relative BiFC positive cells are indicated. *, P

    Techniques Used: Mutagenesis, Activity Assay, Bimolecular Fluorescence Complementation Assay, Transfection, Construct, Quantitation Assay

    Caspase-2-S384E expressing cells fail to cleave MDM2 and show increased polyploidy following cytokinesis block with blebbistatin. GFP-expressing U2OS (sWT) and U2OS- CASP2 -/- expressing GFP (sKO), GFP-caspase-2-C320G (sC320G) or GFP-caspase-2-S384E (sS384 #1 and #2), were treated with DMSO or 50 µM blebbistatin (Bleb) for 48 h and subjected to immunoblot and DNA content analysis. a. Representative immunoblots (of five independent experiments performed) of cell lysates from treated stable cell lines. Antibodies used for immunoblotting are as indicated. β-actin was used as loading control. b. Graph comparing percentage of cells with polyploid ( > 4N) DNA content following Bleb treatment. mean ± SEM; n=5. *, vs. WT + Bleb; *, p
    Figure Legend Snippet: Caspase-2-S384E expressing cells fail to cleave MDM2 and show increased polyploidy following cytokinesis block with blebbistatin. GFP-expressing U2OS (sWT) and U2OS- CASP2 -/- expressing GFP (sKO), GFP-caspase-2-C320G (sC320G) or GFP-caspase-2-S384E (sS384 #1 and #2), were treated with DMSO or 50 µM blebbistatin (Bleb) for 48 h and subjected to immunoblot and DNA content analysis. a. Representative immunoblots (of five independent experiments performed) of cell lysates from treated stable cell lines. Antibodies used for immunoblotting are as indicated. β-actin was used as loading control. b. Graph comparing percentage of cells with polyploid ( > 4N) DNA content following Bleb treatment. mean ± SEM; n=5. *, vs. WT + Bleb; *, p

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

    30) Product Images from "Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome"

    Article Title: Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

    Journal: bioRxiv

    doi: 10.1101/2020.03.30.015370

    Ccr4 does not bind to cellular ubiquitin conjugates under denaturation conditions IP Experiments were done as in Figure 5B except the lysates were subjected to denaturation by addition of 2% SDS, 1% β-mercaptoethanol and heating at 95°C for 5 min. Ccr4-3XFLAG protein was immunoprecipitated as described in methods section. Input levels (left panel) and IP eluate (right panel) were analyzed by western blots using anti-Myc and anti-Flag antibodies. Tpi1 was used as loading control.
    Figure Legend Snippet: Ccr4 does not bind to cellular ubiquitin conjugates under denaturation conditions IP Experiments were done as in Figure 5B except the lysates were subjected to denaturation by addition of 2% SDS, 1% β-mercaptoethanol and heating at 95°C for 5 min. Ccr4-3XFLAG protein was immunoprecipitated as described in methods section. Input levels (left panel) and IP eluate (right panel) were analyzed by western blots using anti-Myc and anti-Flag antibodies. Tpi1 was used as loading control.

    Techniques Used: Immunoprecipitation, Western Blot

    31) Product Images from "In silico design of a T-cell epitope vaccine candidate for parasitic helminth infection"

    Article Title: In silico design of a T-cell epitope vaccine candidate for parasitic helminth infection

    Journal: bioRxiv

    doi: 10.1101/859017

    (A) Inflammatory cytokines production by mouse bone marrow-derived DCs (BMDCs) and mouse bone marrow-derived macrophages (BMDMs) (B) in response to VLPs. BMDCs and BMDMs at 1×10 6 /ml were stimulated in vitro with 10 µg/ml VLPs (HBc-Ag, HBc-H 112-128 , HBc-CBD 1243-1259, HBc-CBD 241-257 , HBc-CLSP 143-158 and HBc-CLSP 398-416 ) and with 50 µg/ml ES and 0.1 µg/ml LPS as positive controls. Unstimulated BMDCs and BMDMs served as negative controls. Supernatants were harvested after 24 hours for IL-6 and TNF α cytokine analyses measured by CBA. The bars represent mean ± SEM. Statistical analyses were carried out using the Kruskal-Wallis test (multiple comparisons). Significant differences between groups are represented by (*P≤0.05) with a line. Chart bars represent BMDCs and BMDMs grown from three individual mice from one representative experiment of two separate experiments. (C) Representative images of fluorescein-conjugated VLPs internalisation in the BMDCs and BMDMs (G). BMDCs and BMDMs at 1×10 6 /ml were incubated with 10 μg/ml fluorescein-conjugated VLP (HBc-Ag and HBc-CLSP 398-416 ) for 24 hours. As a negative control, unstimulated BMDCs and BMDMs were examined. Cell internalisation was determined by Amnis ImageStreamX cytometer compared to unstimulated BMDCs and BMDMs. Images shown, from left to right, show individual Brightfield images (BF) in the white channel, fluorescent-labelled stimulus (FITC) in the green channel and the combination of both BF/FITC merged channels. The internalisation mean absolute deviation (MAD) is included above its images. The positive MAD value represents internalisation, and negative values represent poor internalisation. (D) Representative images of fluorescein-conjugated VLPs co-localization in the BMDCs and BMDMs (H). BMDCs and BMDMs at 1×10 6 /ml were stained with Lysotracker to visualise the cellular lysosome compartment and subsequently stimulated with 10 μg/ml fluorescein-conjugated VLP (HBc-Ag, and HBc-CLSP 398-416 ) for 24 hours. As a negative control, unstimulated BMDCs and BMDMs were examined. Intracellular co-localization was determined by Amnis ImageStreamX cytometer. Images shown, from left to right, show individual Brightfield images (BF) in the white channel, fluorescent-labelled stimulus (FITC) in the green channel, stained lysosome (Lysotracker) in the red channel and the combination of both FITC/ Lysotracker merged channels. The Similarity bright detail score (SBDS) from the IDEAS quantitative co-localization analysis is included above its image. SBDS values around 1 represent co-localization, and 0 values represent poor co-localization. Scale bars represent 10 μm.
    Figure Legend Snippet: (A) Inflammatory cytokines production by mouse bone marrow-derived DCs (BMDCs) and mouse bone marrow-derived macrophages (BMDMs) (B) in response to VLPs. BMDCs and BMDMs at 1×10 6 /ml were stimulated in vitro with 10 µg/ml VLPs (HBc-Ag, HBc-H 112-128 , HBc-CBD 1243-1259, HBc-CBD 241-257 , HBc-CLSP 143-158 and HBc-CLSP 398-416 ) and with 50 µg/ml ES and 0.1 µg/ml LPS as positive controls. Unstimulated BMDCs and BMDMs served as negative controls. Supernatants were harvested after 24 hours for IL-6 and TNF α cytokine analyses measured by CBA. The bars represent mean ± SEM. Statistical analyses were carried out using the Kruskal-Wallis test (multiple comparisons). Significant differences between groups are represented by (*P≤0.05) with a line. Chart bars represent BMDCs and BMDMs grown from three individual mice from one representative experiment of two separate experiments. (C) Representative images of fluorescein-conjugated VLPs internalisation in the BMDCs and BMDMs (G). BMDCs and BMDMs at 1×10 6 /ml were incubated with 10 μg/ml fluorescein-conjugated VLP (HBc-Ag and HBc-CLSP 398-416 ) for 24 hours. As a negative control, unstimulated BMDCs and BMDMs were examined. Cell internalisation was determined by Amnis ImageStreamX cytometer compared to unstimulated BMDCs and BMDMs. Images shown, from left to right, show individual Brightfield images (BF) in the white channel, fluorescent-labelled stimulus (FITC) in the green channel and the combination of both BF/FITC merged channels. The internalisation mean absolute deviation (MAD) is included above its images. The positive MAD value represents internalisation, and negative values represent poor internalisation. (D) Representative images of fluorescein-conjugated VLPs co-localization in the BMDCs and BMDMs (H). BMDCs and BMDMs at 1×10 6 /ml were stained with Lysotracker to visualise the cellular lysosome compartment and subsequently stimulated with 10 μg/ml fluorescein-conjugated VLP (HBc-Ag, and HBc-CLSP 398-416 ) for 24 hours. As a negative control, unstimulated BMDCs and BMDMs were examined. Intracellular co-localization was determined by Amnis ImageStreamX cytometer. Images shown, from left to right, show individual Brightfield images (BF) in the white channel, fluorescent-labelled stimulus (FITC) in the green channel, stained lysosome (Lysotracker) in the red channel and the combination of both FITC/ Lysotracker merged channels. The Similarity bright detail score (SBDS) from the IDEAS quantitative co-localization analysis is included above its image. SBDS values around 1 represent co-localization, and 0 values represent poor co-localization. Scale bars represent 10 μm.

    Techniques Used: Derivative Assay, In Vitro, Crocin Bleaching Assay, Mouse Assay, Incubation, Negative Control, Cytometry, Staining

    32) Product Images from "Dopaminergic Neurodegeneration Induced by Parkinson’s Disease-Linked G2019S LRRK2 is Dependent on Kinase and GTPase Activity"

    Article Title: Dopaminergic Neurodegeneration Induced by Parkinson’s Disease-Linked G2019S LRRK2 is Dependent on Kinase and GTPase Activity

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879759

    Ad5-G2019S-LRRK2 induces dopaminergic neurodegeneration in a GTPase-dependent manner in adult rat brain. A) Immunohistochemistry indicating Ad5 capsid (anti-Hexon) in the ipsilateral rat striatum at 42 days post-injection of Ad5-LRRK2 vectors. Scale bars: 500 μm. B) Immunohistochemistry showing nigral dopaminergic neurons (anti-TH antibody) at 42 days post-delivery. All sections were counterstained with Cresyl violet. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 42 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 8 animals/vector), * P
    Figure Legend Snippet: Ad5-G2019S-LRRK2 induces dopaminergic neurodegeneration in a GTPase-dependent manner in adult rat brain. A) Immunohistochemistry indicating Ad5 capsid (anti-Hexon) in the ipsilateral rat striatum at 42 days post-injection of Ad5-LRRK2 vectors. Scale bars: 500 μm. B) Immunohistochemistry showing nigral dopaminergic neurons (anti-TH antibody) at 42 days post-delivery. All sections were counterstained with Cresyl violet. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 42 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 8 animals/vector), * P

    Techniques Used: Immunohistochemistry, Injection, Plasmid Preparation

    Evaluation of Ad5 vector infectivity in rat brain via distinct delivery paradigms. A) Validation of new high-titer Ad5-LRRK2 (WT, G2019S and G2019S/K1906M) vectors and LRRK2 phosphorylation in SH-SY5Y cells. Cells were transduced with increasing viral particles (vp) of each Ad5-LRRK2 vector (0.5, 1 or 5.0 x 10 10 vp/well). Western blot analysis of cell extracts with FLAG antibody (transduced human LRRK2) or antibodies to detect LRRK2 autophosphorylation (pSer1292) or constitutive phosphorylation (pSer935, pSer910) sites to detect a titer-dependent increase in human LRRK2 levels/activity. Ponceau S stain was used as a protein loading control. Molecular mass markers are indicated in kilodaltons. B) Evaluation of Ad5-LRRK2 vectors following intrastriatal delivery with increasing viral titer (0.5, 1 or 1.5 x 10 10 vp/site, at 2.5 μl/site) at six distinct injection sites. Immunohistochemistry showing dose-dependent expression of human LRRK2-WT (anti-FLAG antibody) in the ipsilateral rat striatum and substantia nigra at 10 days post-injection. Scale bars: 500 μm. C) Evaluation of Ad5-LRRK2 vectors following intranigral delivery with increasing viral titer (0.5, 1 or 1.5 x 10 10 vp/site, in 2.5 μl) at a single injection site. Immunohistochemistry showing dose-dependent expression of human LRRK2-WT (anti-FLAG antibody) in rat substantia nigra at 10 days post-injection. Scale bars: 500 μm.
    Figure Legend Snippet: Evaluation of Ad5 vector infectivity in rat brain via distinct delivery paradigms. A) Validation of new high-titer Ad5-LRRK2 (WT, G2019S and G2019S/K1906M) vectors and LRRK2 phosphorylation in SH-SY5Y cells. Cells were transduced with increasing viral particles (vp) of each Ad5-LRRK2 vector (0.5, 1 or 5.0 x 10 10 vp/well). Western blot analysis of cell extracts with FLAG antibody (transduced human LRRK2) or antibodies to detect LRRK2 autophosphorylation (pSer1292) or constitutive phosphorylation (pSer935, pSer910) sites to detect a titer-dependent increase in human LRRK2 levels/activity. Ponceau S stain was used as a protein loading control. Molecular mass markers are indicated in kilodaltons. B) Evaluation of Ad5-LRRK2 vectors following intrastriatal delivery with increasing viral titer (0.5, 1 or 1.5 x 10 10 vp/site, at 2.5 μl/site) at six distinct injection sites. Immunohistochemistry showing dose-dependent expression of human LRRK2-WT (anti-FLAG antibody) in the ipsilateral rat striatum and substantia nigra at 10 days post-injection. Scale bars: 500 μm. C) Evaluation of Ad5-LRRK2 vectors following intranigral delivery with increasing viral titer (0.5, 1 or 1.5 x 10 10 vp/site, in 2.5 μl) at a single injection site. Immunohistochemistry showing dose-dependent expression of human LRRK2-WT (anti-FLAG antibody) in rat substantia nigra at 10 days post-injection. Scale bars: 500 μm.

    Techniques Used: Plasmid Preparation, Infection, Transduction, Western Blot, Activity Assay, Staining, Injection, Immunohistochemistry, Expressing

    Evaluation of Ad5-LRRK2-G2019S vectors with genetic disruption of GTP-binding or GTP hydrolysis in cells and rat brain. A) Validation of high-titer Ad5-LRRK2 (G2019S, G2019S/R1398L, G2019S/R1398L/T1343V or G2019S/T1348N) vectors and LRRK2 phosphorylation in SH-SY5Y cells. Cells were transduced with increasing viral particles (vp) of each Ad5-LRRK2 vector (0.5, 1 or 5.0 x 10 10 vp/well). Western blot analysis of cell extracts with FLAG antibody (transduced human LRRK2) or antibodies to detect LRRK2 autophosphorylation (pSer1292) or constitutive phosphorylation (pSer935, pSer910) sites to detect a titer-dependent increase in human LRRK2 levels/activity. β-tubulin was used as protein loading control. B) Ipsilateral striatum was harvested at 10 days after unilateral intrastriatal delivery of Ad5-LRRK2 vectors and subjected to sequential detergent extraction in 1% Triton X100 and 2% SDS. Striatal fractions were analyzed by Western blot analysis ( n = 3 animals/vector) indicating human LRRK2 variant levels (anti-FLAG antibody). Expression of dynamin-I (DnmI) was used as loading control. NI, non-injected striatum. C) Densitometric analysis of human LRRK2 variant levels (FLAG) normalized to Dnm 1 levels in each detergent fraction. Bars represent the mean ± SEM ( n = 3 animals/vector). n.s , non-significant by one-way ANOVA with Bonferroni’s multiple comparisons. D) Ad5-LRRK2 (G2019S, G2019S/R1398L, G2019S/R1398L/T1343V, G2019S/T1348N) vectors were unilaterally delivered at six injection sites (1.5 x 10 10 vp/site, in 2.5 μl) in the rat striatum. Immunohistochemistry indicates human LRRK2 variant (anti-FLAG) expression in the ipsilateral striatum and substantia nigra that persists up to 42 days post-injection. Images indicate low and high magnification for each vector and brain region. Scale bars: 500 μm.
    Figure Legend Snippet: Evaluation of Ad5-LRRK2-G2019S vectors with genetic disruption of GTP-binding or GTP hydrolysis in cells and rat brain. A) Validation of high-titer Ad5-LRRK2 (G2019S, G2019S/R1398L, G2019S/R1398L/T1343V or G2019S/T1348N) vectors and LRRK2 phosphorylation in SH-SY5Y cells. Cells were transduced with increasing viral particles (vp) of each Ad5-LRRK2 vector (0.5, 1 or 5.0 x 10 10 vp/well). Western blot analysis of cell extracts with FLAG antibody (transduced human LRRK2) or antibodies to detect LRRK2 autophosphorylation (pSer1292) or constitutive phosphorylation (pSer935, pSer910) sites to detect a titer-dependent increase in human LRRK2 levels/activity. β-tubulin was used as protein loading control. B) Ipsilateral striatum was harvested at 10 days after unilateral intrastriatal delivery of Ad5-LRRK2 vectors and subjected to sequential detergent extraction in 1% Triton X100 and 2% SDS. Striatal fractions were analyzed by Western blot analysis ( n = 3 animals/vector) indicating human LRRK2 variant levels (anti-FLAG antibody). Expression of dynamin-I (DnmI) was used as loading control. NI, non-injected striatum. C) Densitometric analysis of human LRRK2 variant levels (FLAG) normalized to Dnm 1 levels in each detergent fraction. Bars represent the mean ± SEM ( n = 3 animals/vector). n.s , non-significant by one-way ANOVA with Bonferroni’s multiple comparisons. D) Ad5-LRRK2 (G2019S, G2019S/R1398L, G2019S/R1398L/T1343V, G2019S/T1348N) vectors were unilaterally delivered at six injection sites (1.5 x 10 10 vp/site, in 2.5 μl) in the rat striatum. Immunohistochemistry indicates human LRRK2 variant (anti-FLAG) expression in the ipsilateral striatum and substantia nigra that persists up to 42 days post-injection. Images indicate low and high magnification for each vector and brain region. Scale bars: 500 μm.

    Techniques Used: Binding Assay, Transduction, Plasmid Preparation, Western Blot, Activity Assay, Variant Assay, Expressing, Injection, Immunohistochemistry

    In-diet pharmacological kinase inhibition (PF-360) markedly destabilizes human G2019S-LRRK2 protein in rat brain. A) Ad5-G2019S-LRRK2 vectors were unilaterally delivered at six distinct locations (1.5 x 10 10 vp/site, in 2.5 μl) in the rat striatum. Rats were fed with control (vehicle) or PF-360 (35 or 175 mg/kg) chow at day 7 post-injection for 7 consecutive days. Brain, peripheral tissues (lung, kidney, spleen) and PBMCs were collected at 14 days post-injection and subjected to sequential extraction in buffers containing 1% Triton X100 (Triton-soluble fraction) and 2% SDS (SDS-soluble fraction). B-C) Western blot analysis of ipsilateral striatum extracts indicating human G2019S-LRRK2 levels (anti-FLAG antibody), or contralateral striatum extracts indicating phosphorylated (pSer935) and total (MJFF2/c41-2) endogenous LRRK2 levels in rats fed with vehicle or PF-360 chow at B) 35 or C) 175 mg/kg chow. Dynamin I (DnmI) or β-tubulin were used as loading controls for normalization. NI, non-injected striatum. Densitometric quantitation is shown for human G2019S-LRRK2 (FLAG) levels in each detergent fraction, and endogenous pSer935-LRRK2 or total LRRK2 levels. Bars represent the mean ± SEM ( n = 5 animals/vector), * P
    Figure Legend Snippet: In-diet pharmacological kinase inhibition (PF-360) markedly destabilizes human G2019S-LRRK2 protein in rat brain. A) Ad5-G2019S-LRRK2 vectors were unilaterally delivered at six distinct locations (1.5 x 10 10 vp/site, in 2.5 μl) in the rat striatum. Rats were fed with control (vehicle) or PF-360 (35 or 175 mg/kg) chow at day 7 post-injection for 7 consecutive days. Brain, peripheral tissues (lung, kidney, spleen) and PBMCs were collected at 14 days post-injection and subjected to sequential extraction in buffers containing 1% Triton X100 (Triton-soluble fraction) and 2% SDS (SDS-soluble fraction). B-C) Western blot analysis of ipsilateral striatum extracts indicating human G2019S-LRRK2 levels (anti-FLAG antibody), or contralateral striatum extracts indicating phosphorylated (pSer935) and total (MJFF2/c41-2) endogenous LRRK2 levels in rats fed with vehicle or PF-360 chow at B) 35 or C) 175 mg/kg chow. Dynamin I (DnmI) or β-tubulin were used as loading controls for normalization. NI, non-injected striatum. Densitometric quantitation is shown for human G2019S-LRRK2 (FLAG) levels in each detergent fraction, and endogenous pSer935-LRRK2 or total LRRK2 levels. Bars represent the mean ± SEM ( n = 5 animals/vector), * P

    Techniques Used: Inhibition, Injection, Western Blot, Quantitation Assay, Plasmid Preparation

    Intranigral delivery of Ad5-LRRK2-G2019S induces dopaminergic neurodegeneration in a kinase-dependent manner in adult rat brain. A) Ad5-LRRK2 (WT, G2019S or G2019S/K1906M) vectors were unilaterally delivered at a single site (1.5 x 10 10 vp/site, in 2.5 μl) in the rat substantia nigra. Immunohistochemistry reveals expression of eGFP (anti-GFP antibody) or human LRRK2 variants (anti-FLAG antibody) that persists up to 21 days post-injection. Images indicate low and high magnification for each vector. Scale bars: 500 μm. B) Immunohistochemistry revealing dopaminergic neurons (anti-TH antibody) in contralateral and ipsilateral substantia nigra at 21 days post-delivery of Ad5 vectors. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 21 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 7-8 animals/vector), * P
    Figure Legend Snippet: Intranigral delivery of Ad5-LRRK2-G2019S induces dopaminergic neurodegeneration in a kinase-dependent manner in adult rat brain. A) Ad5-LRRK2 (WT, G2019S or G2019S/K1906M) vectors were unilaterally delivered at a single site (1.5 x 10 10 vp/site, in 2.5 μl) in the rat substantia nigra. Immunohistochemistry reveals expression of eGFP (anti-GFP antibody) or human LRRK2 variants (anti-FLAG antibody) that persists up to 21 days post-injection. Images indicate low and high magnification for each vector. Scale bars: 500 μm. B) Immunohistochemistry revealing dopaminergic neurons (anti-TH antibody) in contralateral and ipsilateral substantia nigra at 21 days post-delivery of Ad5 vectors. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 21 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 7-8 animals/vector), * P

    Techniques Used: Immunohistochemistry, Expressing, Injection, Plasmid Preparation

    Intrastriatal delivery of Ad5-LRRK2-G2019S induces kinase-dependent dopaminergic neurodegeneration in adult rat brain. A) Ad5-LRRK2 (WT, G2019S and G2019S/K1906M) vectors were unilaterally delivered at six injection sites (1.5 x 10 10 vp/site, in 2.5 μl) to the rat striatum. Immunohistochemistry reveals expression of eGFP (anti-GFP antibody) or human LRRK2 variants (anti-FLAG antibody) in the ipsilateral striatum and nigra that persists up to 42 days post-injection. Images indicate low and high magnification for each vector and brain region. Scale bars: 500 μm. B) Immunohistochemistry revealing dopaminergic neurons (anti-TH antibody) in contralateral and ipsilateral substantia nigra at 42 days post-delivery of Ad5 vectors. All sections were counterstained with Cresyl violet. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 42 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 8 animals/vector), * P
    Figure Legend Snippet: Intrastriatal delivery of Ad5-LRRK2-G2019S induces kinase-dependent dopaminergic neurodegeneration in adult rat brain. A) Ad5-LRRK2 (WT, G2019S and G2019S/K1906M) vectors were unilaterally delivered at six injection sites (1.5 x 10 10 vp/site, in 2.5 μl) to the rat striatum. Immunohistochemistry reveals expression of eGFP (anti-GFP antibody) or human LRRK2 variants (anti-FLAG antibody) in the ipsilateral striatum and nigra that persists up to 42 days post-injection. Images indicate low and high magnification for each vector and brain region. Scale bars: 500 μm. B) Immunohistochemistry revealing dopaminergic neurons (anti-TH antibody) in contralateral and ipsilateral substantia nigra at 42 days post-delivery of Ad5 vectors. All sections were counterstained with Cresyl violet. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 42 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 8 animals/vector), * P

    Techniques Used: Injection, Immunohistochemistry, Expressing, Plasmid Preparation

    Pilot studies evaluating role of kinase activity using new Ad5-LRRK2-G2019S vectors. A) Validation of Ad5 vectors in SH-SY5Y cells. Cells were transduced with increasing viral particles (vp) of each Ad5-eGFP or Ad5-LRRK2 vector (0.5, 1 or 1.5 x 10 10 vp/well). Western blot analysis of cell extracts with GFP or FLAG antibody to detect a titer-dependent increase in human LRRK2 levels. Ponceau S stain was used as a protein loading control. Molecular mass markers are indicated in kilodaltons. B) Ad5-eGFP and Ad5-LRRK2 (G2019S, G2019S/D1994N or G2019S/K1906M) vectors were delivered at a single injection site (4.5 x 10 9 vp/site, in 2.5 µl) in the ipsilateral substantia nigra pars compacta of rats, as indicated (red arrow). Immunohistochemistry for eGFP (anti-GFP antibody) or human LRRK2 variants (anti-FLAG antibody) in the ipsilateral substantia nigra at 10 days post-injection. Images indicate low and high magnification for each vector. Scale bar: 500 μm. C) Immunohistochemistry revealing dopaminergic neurons (anti-TH antibody) in contralateral and ipsilateral substantia nigra at 21 days post-delivery of Ad5 vectors. All sections were counterstained with Cresyl violet. D) Analysis of neurodegeneration in rat substantia nigra at 21 days. TH-positive dopaminergic neuron number in the substantia nigra assessed by unbiased stereology. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 4 animals/vector), * P
    Figure Legend Snippet: Pilot studies evaluating role of kinase activity using new Ad5-LRRK2-G2019S vectors. A) Validation of Ad5 vectors in SH-SY5Y cells. Cells were transduced with increasing viral particles (vp) of each Ad5-eGFP or Ad5-LRRK2 vector (0.5, 1 or 1.5 x 10 10 vp/well). Western blot analysis of cell extracts with GFP or FLAG antibody to detect a titer-dependent increase in human LRRK2 levels. Ponceau S stain was used as a protein loading control. Molecular mass markers are indicated in kilodaltons. B) Ad5-eGFP and Ad5-LRRK2 (G2019S, G2019S/D1994N or G2019S/K1906M) vectors were delivered at a single injection site (4.5 x 10 9 vp/site, in 2.5 µl) in the ipsilateral substantia nigra pars compacta of rats, as indicated (red arrow). Immunohistochemistry for eGFP (anti-GFP antibody) or human LRRK2 variants (anti-FLAG antibody) in the ipsilateral substantia nigra at 10 days post-injection. Images indicate low and high magnification for each vector. Scale bar: 500 μm. C) Immunohistochemistry revealing dopaminergic neurons (anti-TH antibody) in contralateral and ipsilateral substantia nigra at 21 days post-delivery of Ad5 vectors. All sections were counterstained with Cresyl violet. D) Analysis of neurodegeneration in rat substantia nigra at 21 days. TH-positive dopaminergic neuron number in the substantia nigra assessed by unbiased stereology. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 4 animals/vector), * P

    Techniques Used: Activity Assay, Transduction, Plasmid Preparation, Western Blot, Staining, Injection, Immunohistochemistry

    Pharmacological kinase inhibition (PF-360) protects against dopaminergic neurodegeneration induced by human G2019S LRRK2. A) Ad5-G2019S-LRRK2 vectors were unilaterally delivered at six distinct locations (1.5 x 10 10 vp/site, in 2.5 μl) to the rat striatum. Rats were fed continuously with control (vehicle) or PF-360 (175 mg/kg) chow from days 7 to 42 post-injection. Brain tissues were harvested at 42 days post-injection and subjected to immunohistochemical analysis. B) Immunohistochemistry reveals expression of human LRRK2 (anti-FLAG antibody) in striatum and nigra, or nigral dopaminergic neurons (anti-TH antibody), of rats at 42 days post-injection. Images indicate low and high magnification for each group and brain region. TH sections were counterstained with Cresyl violet. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 42 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 12 animals/group), * P
    Figure Legend Snippet: Pharmacological kinase inhibition (PF-360) protects against dopaminergic neurodegeneration induced by human G2019S LRRK2. A) Ad5-G2019S-LRRK2 vectors were unilaterally delivered at six distinct locations (1.5 x 10 10 vp/site, in 2.5 μl) to the rat striatum. Rats were fed continuously with control (vehicle) or PF-360 (175 mg/kg) chow from days 7 to 42 post-injection. Brain tissues were harvested at 42 days post-injection and subjected to immunohistochemical analysis. B) Immunohistochemistry reveals expression of human LRRK2 (anti-FLAG antibody) in striatum and nigra, or nigral dopaminergic neurons (anti-TH antibody), of rats at 42 days post-injection. Images indicate low and high magnification for each group and brain region. TH sections were counterstained with Cresyl violet. Scale bars: 500 μm. C) Unbiased stereological analysis of TH-positive dopaminergic and total Nissl-positive neuron number in the substantia nigra at 42 days post-injection. Bars represent % neuronal loss in the injected ipsilateral nigra relative to the contralateral nigra (mean ± SEM, n = 12 animals/group), * P

    Techniques Used: Inhibition, Injection, Immunohistochemistry, Expressing

    33) Product Images from "A Panel of Diverse Assays to Interrogate the Interaction between Glucokinase and Glucokinase Regulatory Protein, Two Vital Proteins in Human Disease"

    Article Title: A Panel of Diverse Assays to Interrogate the Interaction between Glucokinase and Glucokinase Regulatory Protein, Two Vital Proteins in Human Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089335

    Parallel assay development strategy to interrogate recombinant GCK and GKRP. Human GCK and GKRP were affinity-purified using GST and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.
    Figure Legend Snippet: Parallel assay development strategy to interrogate recombinant GCK and GKRP. Human GCK and GKRP were affinity-purified using GST and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Glo™ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Glo™ Reagent; Reagent 2: Kinase Detection Reagent.

    Techniques Used: Recombinant, Affinity Purification, Luciferase

    34) Product Images from "A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver"

    Article Title: A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver

    Journal: eLife

    doi: 10.7554/eLife.09003

    Effects of TA, other candidate hits, and published sumoylation inhibitors in an IVS assay of full-length hLRH1. ( A ) Dose-dependent inhibition of full-length (FL)-hLRH-1 by TA compared to other top candidate hits from primary screen as assayed by IVS. IVS assay and immunoblotting conditions used to detect hLRH-1 species are described in Materials and Methods. Sumoylated hLRH-1 (1x, 2x, 3x) and unmodified LRH-1 (LRH-1) species are indicated by arrows. ( B ) IVS assays of FL-hLRH-1 were performed with increasing concentrations of TA are shown (left panel) and plotted as normalized values in graph (right panel). Effects of two other published sumoylation inhibitors, 2-D08 and GA are also shown in graph. IVS data was normalized to DMSO control for each compound, and then plotted as percent conversion per log 10 [µM] concentration. Curve fitting of data is described in ‘Materials and methods’. GA: Ginkgolic acid; IVS: In vitro sumoylation; TA: Tannic acid. DOI: http://dx.doi.org/10.7554/eLife.09003.013
    Figure Legend Snippet: Effects of TA, other candidate hits, and published sumoylation inhibitors in an IVS assay of full-length hLRH1. ( A ) Dose-dependent inhibition of full-length (FL)-hLRH-1 by TA compared to other top candidate hits from primary screen as assayed by IVS. IVS assay and immunoblotting conditions used to detect hLRH-1 species are described in Materials and Methods. Sumoylated hLRH-1 (1x, 2x, 3x) and unmodified LRH-1 (LRH-1) species are indicated by arrows. ( B ) IVS assays of FL-hLRH-1 were performed with increasing concentrations of TA are shown (left panel) and plotted as normalized values in graph (right panel). Effects of two other published sumoylation inhibitors, 2-D08 and GA are also shown in graph. IVS data was normalized to DMSO control for each compound, and then plotted as percent conversion per log 10 [µM] concentration. Curve fitting of data is described in ‘Materials and methods’. GA: Ginkgolic acid; IVS: In vitro sumoylation; TA: Tannic acid. DOI: http://dx.doi.org/10.7554/eLife.09003.013

    Techniques Used: Inhibition, Concentration Assay, In Vitro

    Human LRH-1 transcripts and protein are expressed in liver after AAV8-TBG viral infection. ( A ) Relative expression of hLRH-1 or endogenous mLrh-1 transcripts in mouse liver 14 days post-infection with either AAV8-eGFP (GFP) or AAV8 hLRH-1 (hLRH-1) at a vector genome titer of 1 x 10 11 (genome copies/ml or GC/ml). ( B ) Human LRH-1 detected by anti-Flag antibody in heart and liver tissue collected from mice expressing Flag-hLRH-1 (hLRH-1) or eGFP as described in ‘Materials and methods’. Human Flag-tagged LRH-1 protein expressed in HepG2 cells is indicated with arrow (far left lane) and loading controls for each sample (Gapdh). DOI: http://dx.doi.org/10.7554/eLife.09003.006
    Figure Legend Snippet: Human LRH-1 transcripts and protein are expressed in liver after AAV8-TBG viral infection. ( A ) Relative expression of hLRH-1 or endogenous mLrh-1 transcripts in mouse liver 14 days post-infection with either AAV8-eGFP (GFP) or AAV8 hLRH-1 (hLRH-1) at a vector genome titer of 1 x 10 11 (genome copies/ml or GC/ml). ( B ) Human LRH-1 detected by anti-Flag antibody in heart and liver tissue collected from mice expressing Flag-hLRH-1 (hLRH-1) or eGFP as described in ‘Materials and methods’. Human Flag-tagged LRH-1 protein expressed in HepG2 cells is indicated with arrow (far left lane) and loading controls for each sample (Gapdh). DOI: http://dx.doi.org/10.7554/eLife.09003.006

    Techniques Used: Infection, Expressing, Plasmid Preparation, Mouse Assay

    35) Product Images from "Structural basis for recognition of the Sec4 Rab GTPase by its effector, the Lgl/tomosyn homologue, Sro7"

    Article Title: Structural basis for recognition of the Sec4 Rab GTPase by its effector, the Lgl/tomosyn homologue, Sro7

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-04-0228

    The interaction between Sro7 and the yeast Rab GTPase Sec4 is specific and GTP dependent. Soluble Sro7 was tested for binding to eight representatives of the yeast Rab GTPase family immobilized on glutathione Sepharose beads after exchange with GTPγS, GDP, or no nucleotide. Coomassie gel compares the amounts of Rab GTPases used in the in vitro binding. Quantitation is based on three independent experiments.
    Figure Legend Snippet: The interaction between Sro7 and the yeast Rab GTPase Sec4 is specific and GTP dependent. Soluble Sro7 was tested for binding to eight representatives of the yeast Rab GTPase family immobilized on glutathione Sepharose beads after exchange with GTPγS, GDP, or no nucleotide. Coomassie gel compares the amounts of Rab GTPases used in the in vitro binding. Quantitation is based on three independent experiments.

    Techniques Used: Binding Assay, In Vitro, Quantitation Assay

    Computational docking studies extracted interacting elements from the best-scoring complexes of Sro7 and Sec4-GTP to produce four models. (A) Surface-filling model of Sro7 with an overlay of the four Sec4 docking arrangements. The Sro7 N-terminal propeller is shown in blue, the C-terminal propeller is in green, and the C-terminal tail is in light pink. The Sec4 ribbon diagrams (models A–D) are colored in yellow, pink, red and purple, respectively. (B) Ribbon diagram of Sro7, with new mutations at the Sro7–Sec4 interface marked in orange. Original mutations are marked in red. (C) Purified wild-type Sro7 or Sro7 mutants were tested for binding to GST-Sec4 after exchange with either GTPγS or GDP. Western blot and quantitation from four independent experiments. The mutant strain sec15-1 was transformed with a plasmid ( CEN ) expressing SRO7 , the novel discriminatory mutants, or vector only. Three independent transformants were picked into microtiter wells and transferred to YPD medium at 25 and 37°C.
    Figure Legend Snippet: Computational docking studies extracted interacting elements from the best-scoring complexes of Sro7 and Sec4-GTP to produce four models. (A) Surface-filling model of Sro7 with an overlay of the four Sec4 docking arrangements. The Sro7 N-terminal propeller is shown in blue, the C-terminal propeller is in green, and the C-terminal tail is in light pink. The Sec4 ribbon diagrams (models A–D) are colored in yellow, pink, red and purple, respectively. (B) Ribbon diagram of Sro7, with new mutations at the Sro7–Sec4 interface marked in orange. Original mutations are marked in red. (C) Purified wild-type Sro7 or Sro7 mutants were tested for binding to GST-Sec4 after exchange with either GTPγS or GDP. Western blot and quantitation from four independent experiments. The mutant strain sec15-1 was transformed with a plasmid ( CEN ) expressing SRO7 , the novel discriminatory mutants, or vector only. Three independent transformants were picked into microtiter wells and transferred to YPD medium at 25 and 37°C.

    Techniques Used: Purification, Binding Assay, Western Blot, Quantitation Assay, Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing

    Biochemical screen identifies two Sro7 mutants deficient in binding to Sec4-GTP. (A) Schematic of Sro7 showing the N-terminal propeller in blue, the C-terminal propeller in green, and the autoinhibitory tail in light pink. Sites of charge-reversal mutations are indicated in fuchsia. (B) Surface-filling models of Sro7 showing the 12 residues subjected to mutation in fuchsia. (C) Coomassie gel of purified Sro7 and the Sro7 charge-reversal mutant proteins. Quantitation of Sro7 and Sro7 mutant protein binding to GST, GST-Sec9, or GST-Sec4 previously loaded with either GTPγS or GDP. Binding was expressed as a percentage, with wild-type Sro7 binding set to 100%. Quantitation in each graph was based on four independent experiments. (D) The mutant strain sec15-1 was transformed with a plasmid ( CEN ) expressing SRO7 , the charge-reversal mutants, or vector only. Three independent transformants were picked into microtiter wells and transferred to YPD medium at 25 and 37°C. (E) Wild-type and SRO7 mutants unable to bind Sec4 were integrated as the sole source at the SRO7 locus. Four independent colonies were picked into microtiter wells and transferred to YPD medium at 37, 25, and 17°C.
    Figure Legend Snippet: Biochemical screen identifies two Sro7 mutants deficient in binding to Sec4-GTP. (A) Schematic of Sro7 showing the N-terminal propeller in blue, the C-terminal propeller in green, and the autoinhibitory tail in light pink. Sites of charge-reversal mutations are indicated in fuchsia. (B) Surface-filling models of Sro7 showing the 12 residues subjected to mutation in fuchsia. (C) Coomassie gel of purified Sro7 and the Sro7 charge-reversal mutant proteins. Quantitation of Sro7 and Sro7 mutant protein binding to GST, GST-Sec9, or GST-Sec4 previously loaded with either GTPγS or GDP. Binding was expressed as a percentage, with wild-type Sro7 binding set to 100%. Quantitation in each graph was based on four independent experiments. (D) The mutant strain sec15-1 was transformed with a plasmid ( CEN ) expressing SRO7 , the charge-reversal mutants, or vector only. Three independent transformants were picked into microtiter wells and transferred to YPD medium at 25 and 37°C. (E) Wild-type and SRO7 mutants unable to bind Sec4 were integrated as the sole source at the SRO7 locus. Four independent colonies were picked into microtiter wells and transferred to YPD medium at 37, 25, and 17°C.

    Techniques Used: Binding Assay, Mutagenesis, Purification, Quantitation Assay, Protein Binding, Transformation Assay, Plasmid Preparation, Expressing

    36) Product Images from "A redox mechanism underlying nucleolar stress sensing by nucleophosmin"

    Article Title: A redox mechanism underlying nucleolar stress sensing by nucleophosmin

    Journal: Nature Communications

    doi: 10.1038/ncomms13599

    Cys 275 oxidation is responsible for NPM1 translocation. ( a – c ) Translocation of mCherry-NPM1 WT and EGFP-NPM1 mutants in identical cells upon H 2 O 2 (500 μM) exposure, visualized by live-cell imaging for 20 min. ( d ) Localization of various FLAG-NPM1 mutants in unstressed HeLa cells. Scale bars, 5 μm. 3C/S, C21/104/275S.
    Figure Legend Snippet: Cys 275 oxidation is responsible for NPM1 translocation. ( a – c ) Translocation of mCherry-NPM1 WT and EGFP-NPM1 mutants in identical cells upon H 2 O 2 (500 μM) exposure, visualized by live-cell imaging for 20 min. ( d ) Localization of various FLAG-NPM1 mutants in unstressed HeLa cells. Scale bars, 5 μm. 3C/S, C21/104/275S.

    Techniques Used: Translocation Assay, Live Cell Imaging

    The presence of nucleoplasmic NPM1 is a prerequisite for stress-induced p53 activation. ( a ) FLAG-NPM1 WT and the mutants WW and C275S were added back to NPM1-silenced cells before treatment with Act.D. Co-IP assays were performed using antibody against HDM2 and western blottings were performed using antibodies as indicated. ( b ) Localization of exogenous HA-ARF and endogenous NPM1 after Act.D treatment, from respective cells on same coverslip. Nucleolar/nucleoplasmic RFI ratio of NPM1 ( n =29) and ARF ( n =41) were displayed. Mean±s.e.m. Unpaired t -test. *** P
    Figure Legend Snippet: The presence of nucleoplasmic NPM1 is a prerequisite for stress-induced p53 activation. ( a ) FLAG-NPM1 WT and the mutants WW and C275S were added back to NPM1-silenced cells before treatment with Act.D. Co-IP assays were performed using antibody against HDM2 and western blottings were performed using antibodies as indicated. ( b ) Localization of exogenous HA-ARF and endogenous NPM1 after Act.D treatment, from respective cells on same coverslip. Nucleolar/nucleoplasmic RFI ratio of NPM1 ( n =29) and ARF ( n =41) were displayed. Mean±s.e.m. Unpaired t -test. *** P

    Techniques Used: Activation Assay, Activated Clotting Time Assay, Co-Immunoprecipitation Assay, Western Blot

    NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml −1 ) and DNase I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P
    Figure Legend Snippet: NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml −1 ) and DNase I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P

    Techniques Used: Immunoprecipitation, Chromatin Immunoprecipitation

    NPM1 is S -glutathionylated at Cys 275 under stress. ( a ) Non-reduced co-IP assay. After gradient doses of H 2 O 2 (500 μM, 1 mM) treatments±antioxidant NAC (5 mM) pretreatment, S -glutathionylated NPM1 in HeLa cells was co-immunoprecipitated and analysed with either anti-GSH or anti-NPM1 antibodies. ( b ) Tandem mass spectrometry (MS/MS) analysis of NPM1 protein derived from non-reduced co-IP. FLAG-NPM1 was transfected to HEK293T cells exposed to H 2 O 2 (500 μM) and immunoprecipitated by anti-FLAG M2 gel. The amino acid sequence of peptides was reconstructed by analysis of the b −ion or y −ion series. The MS/MS spectrum of the m/z y 5 + (972.4026) was corresponding to the NPM1 peptide KNCFR S -glutathionylated at Cys 275 , in comparison with the non-modified spectrum 667.3344 in Supplementary Fig. 4c . ( c ) NPM1 translocation (left) and RFI changes of nucleolar NPM1 (right, n =57 cells) after H 2 O 2 (500 μM) treatment in HeLa cells. GSTP were silenced with two siRNA oligo and the relative GSTP mRNA level were showed (middle, siGSTP-1 and siGSTP-2). Unpaired t -test, ** P
    Figure Legend Snippet: NPM1 is S -glutathionylated at Cys 275 under stress. ( a ) Non-reduced co-IP assay. After gradient doses of H 2 O 2 (500 μM, 1 mM) treatments±antioxidant NAC (5 mM) pretreatment, S -glutathionylated NPM1 in HeLa cells was co-immunoprecipitated and analysed with either anti-GSH or anti-NPM1 antibodies. ( b ) Tandem mass spectrometry (MS/MS) analysis of NPM1 protein derived from non-reduced co-IP. FLAG-NPM1 was transfected to HEK293T cells exposed to H 2 O 2 (500 μM) and immunoprecipitated by anti-FLAG M2 gel. The amino acid sequence of peptides was reconstructed by analysis of the b −ion or y −ion series. The MS/MS spectrum of the m/z y 5 + (972.4026) was corresponding to the NPM1 peptide KNCFR S -glutathionylated at Cys 275 , in comparison with the non-modified spectrum 667.3344 in Supplementary Fig. 4c . ( c ) NPM1 translocation (left) and RFI changes of nucleolar NPM1 (right, n =57 cells) after H 2 O 2 (500 μM) treatment in HeLa cells. GSTP were silenced with two siRNA oligo and the relative GSTP mRNA level were showed (middle, siGSTP-1 and siGSTP-2). Unpaired t -test, ** P

    Techniques Used: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Derivative Assay, Transfection, Sequencing, Modification, Translocation Assay

    NPM1 translocation is required for p53 activation. ( a , b ) Endogenous NPM1 translocation and p53 accumulation cells under nucleolar stress with western blot validation. ( a ) Cells were pretreated with NAC (5 mM) for 4 h before Act.D (8 nM) treatment, or co-treated with DTT (5 mM) and Act.D (8 nM). Nucleolar line profiles of NPM1 in representative cells are shown in Supplementary Fig. 6a . ( b ) NPM1 was silenced with shRNA (shNPM1 5′-untranslated region (UTR)) before Act.D treatment or H 2 O 2 (500 μM). ( c ) Immunofluorescence for endogenous p53 accumulation in NPM1-silenced cells (shNPM1 5′-UTR) that added back FLAG-NPM1 WT, C275S or WW mutants before treatment with Act.D. Line profiles of p53 in representative cells were showed in Supplementary Fig. 6c . ( d ) p53 accumulation after Act.D (left) or H 2 O 2 (right) treatment in NPM1-silenced cells which were added back WT or C275S mutant of NPM1. Endogenous and exogenous FLAG-NPM1 were blotted with anti-NPM1 antibody. Nutlin-3 was used as a positive control for p53 inducer. ( e ) Relative mRNA level changes of p21 and PUMA after Act.D treatments in NPM1-silenced cells (shNPM1 5′-UTR) that added back FLAG-NPM1 WT or C275S mutants. Unpaired t -test. * P
    Figure Legend Snippet: NPM1 translocation is required for p53 activation. ( a , b ) Endogenous NPM1 translocation and p53 accumulation cells under nucleolar stress with western blot validation. ( a ) Cells were pretreated with NAC (5 mM) for 4 h before Act.D (8 nM) treatment, or co-treated with DTT (5 mM) and Act.D (8 nM). Nucleolar line profiles of NPM1 in representative cells are shown in Supplementary Fig. 6a . ( b ) NPM1 was silenced with shRNA (shNPM1 5′-untranslated region (UTR)) before Act.D treatment or H 2 O 2 (500 μM). ( c ) Immunofluorescence for endogenous p53 accumulation in NPM1-silenced cells (shNPM1 5′-UTR) that added back FLAG-NPM1 WT, C275S or WW mutants before treatment with Act.D. Line profiles of p53 in representative cells were showed in Supplementary Fig. 6c . ( d ) p53 accumulation after Act.D (left) or H 2 O 2 (right) treatment in NPM1-silenced cells which were added back WT or C275S mutant of NPM1. Endogenous and exogenous FLAG-NPM1 were blotted with anti-NPM1 antibody. Nutlin-3 was used as a positive control for p53 inducer. ( e ) Relative mRNA level changes of p21 and PUMA after Act.D treatments in NPM1-silenced cells (shNPM1 5′-UTR) that added back FLAG-NPM1 WT or C275S mutants. Unpaired t -test. * P

    Techniques Used: Translocation Assay, Activation Assay, Western Blot, Activated Clotting Time Assay, shRNA, Immunofluorescence, Mutagenesis, Positive Control

    37) Product Images from "Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A"

    Article Title: Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    Journal: Nature Communications

    doi: 10.1038/ncomms10631

    SCOTIN promotes NS5A trafficking to autophagosomes. ( a , b ) Huh-7 cells were transfected with the indicated plasmids or siRNAs for 48 h, and total cell lysates were subjected to immunoblotting using the indicated antibodies. A GST-expressing pEBG ( a ) or LacZ-V5 ( b ) plasmid was included to monitor transfection efficiency. ( c – f ) Huh-7 cells were transfected with the indicated plasmids followed by treatment with MG132 (1 μM; c ), 3-MA (10 mM; d ), BFA (100 μM; e ) or CQ (50 μM; f ) for 12 h. Total cell lysates were subjected to immunoblotting using the indicated antibodies. Distilled water (DW) was used as a control for 3-MA and CQ, and dimethylsulphoxide (DMSO) was used as a control for BFA and MG132. c-MYC was used as a positive control for MG132. The relative ratios (FLAG-NS5A/LacZ-V5) are shown based on the intensity values quantified using the Multigauge programme (Fuji Film). ( g ) Huh-7 cells transfected with the indicated plasmids along with control or ATG7 siRNA. Extracted lysates were subjected to immunoblotting using the indicated antibodies. ( c – g ) To determine the transfection efficiency, LacZ-V5 was co-transfected with the indicated plasmids. ( h – j ) Huh-7 cells were transfected with FLAG-NS5A along with control or SCOTIN siRNA, and then were treated with rapamycin (2 μM) for 6 h. ( h ) Cellular localization of endogenous LC3 (green), FLAG-NS5A (red) and the nucleus (Hoechst) was detected using confocal fluorescence microscopy. A colocalization image was obtained using the Co-localization Image J Plugin. Scale bars, 10 μm. ( i ) Total cell lysates from the same cell populations were subjected to immunoblotting using the indicated antibodies. ( j ) The extent of colocalization of FLAG-NS5A and LC3 in each cell was measured using Pearson's correlation coefficient with the JAcoP Image J Plugin. The coefficient values were plotted using a whiskers box plot. The box extends from the 25th to 75th percentiles, and the error bars depict the minimum and maximum values. Cells transfected with siCON ( N =46) or with siSCOTIN ( N =50) were analysed. The asterisk denotes the P value calculated using the t -test (*** P value
    Figure Legend Snippet: SCOTIN promotes NS5A trafficking to autophagosomes. ( a , b ) Huh-7 cells were transfected with the indicated plasmids or siRNAs for 48 h, and total cell lysates were subjected to immunoblotting using the indicated antibodies. A GST-expressing pEBG ( a ) or LacZ-V5 ( b ) plasmid was included to monitor transfection efficiency. ( c – f ) Huh-7 cells were transfected with the indicated plasmids followed by treatment with MG132 (1 μM; c ), 3-MA (10 mM; d ), BFA (100 μM; e ) or CQ (50 μM; f ) for 12 h. Total cell lysates were subjected to immunoblotting using the indicated antibodies. Distilled water (DW) was used as a control for 3-MA and CQ, and dimethylsulphoxide (DMSO) was used as a control for BFA and MG132. c-MYC was used as a positive control for MG132. The relative ratios (FLAG-NS5A/LacZ-V5) are shown based on the intensity values quantified using the Multigauge programme (Fuji Film). ( g ) Huh-7 cells transfected with the indicated plasmids along with control or ATG7 siRNA. Extracted lysates were subjected to immunoblotting using the indicated antibodies. ( c – g ) To determine the transfection efficiency, LacZ-V5 was co-transfected with the indicated plasmids. ( h – j ) Huh-7 cells were transfected with FLAG-NS5A along with control or SCOTIN siRNA, and then were treated with rapamycin (2 μM) for 6 h. ( h ) Cellular localization of endogenous LC3 (green), FLAG-NS5A (red) and the nucleus (Hoechst) was detected using confocal fluorescence microscopy. A colocalization image was obtained using the Co-localization Image J Plugin. Scale bars, 10 μm. ( i ) Total cell lysates from the same cell populations were subjected to immunoblotting using the indicated antibodies. ( j ) The extent of colocalization of FLAG-NS5A and LC3 in each cell was measured using Pearson's correlation coefficient with the JAcoP Image J Plugin. The coefficient values were plotted using a whiskers box plot. The box extends from the 25th to 75th percentiles, and the error bars depict the minimum and maximum values. Cells transfected with siCON ( N =46) or with siSCOTIN ( N =50) were analysed. The asterisk denotes the P value calculated using the t -test (*** P value

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Positive Control, Fluorescence, Microscopy

    Physical interaction between NS5A and SCOTIN is required for control of degradation. ( a ) HEK293 cells were transfected with GST-NS5A along with an empty (pcDNA3.1-MYC) or SCOTIN-MYC plasmid for 48 h. GST-NS5A was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( b ) Schematic representation of GST-tagged NS5A deletion constructs. ( c ) HEK293 cells were transfected with the indicated plasmids and subjected to a GST pulldown assay. ( d ) Huh-7 cells were transfected with the indicated plasmids for 48 h, followed by immunoblotting analysis using the indicated antibodies. EGFP was used to monitor transfection efficiency. ( e ) An illustration of the truncated SCOTIN constructs is shown. ( f ) Huh-7 cells were transfected with the indicated SCOTIN mutant constructs, and immunofluorescence analysis was performed using LC3 and MYC antibodies, followed by Hoechst staining. Cellular localization of LC3 (green), MYC (red) and the nucleus (blue) was determined using fluorescence microscopy. Representative images are shown. Higher-magnification images are shown in the right corner. Scale bar, 10 μm. ( g ) Western blot analysis of Huh-7 cells transfected with the indicated plasmids. EGFP-N1 was co-transfected to monitor transfection efficiency. ( h ) HEK293 cells were transfected with GST-NS5A along with the indicated plasmids for 48 h. Total cell lysates were incubated with glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( i , j ) Huh-7 cells were transfected with the indicated plasmids followed by HCVcc infection (10 MOI) for 3 days before harvesting. The intracellular HCV RNA levels were determined using RT–qPCR ( i ), and total cell lysates were subjected to immunoblotting using the indicated antibodies ( j ). The bars indicate the mean value±s.d. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. ** P value
    Figure Legend Snippet: Physical interaction between NS5A and SCOTIN is required for control of degradation. ( a ) HEK293 cells were transfected with GST-NS5A along with an empty (pcDNA3.1-MYC) or SCOTIN-MYC plasmid for 48 h. GST-NS5A was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( b ) Schematic representation of GST-tagged NS5A deletion constructs. ( c ) HEK293 cells were transfected with the indicated plasmids and subjected to a GST pulldown assay. ( d ) Huh-7 cells were transfected with the indicated plasmids for 48 h, followed by immunoblotting analysis using the indicated antibodies. EGFP was used to monitor transfection efficiency. ( e ) An illustration of the truncated SCOTIN constructs is shown. ( f ) Huh-7 cells were transfected with the indicated SCOTIN mutant constructs, and immunofluorescence analysis was performed using LC3 and MYC antibodies, followed by Hoechst staining. Cellular localization of LC3 (green), MYC (red) and the nucleus (blue) was determined using fluorescence microscopy. Representative images are shown. Higher-magnification images are shown in the right corner. Scale bar, 10 μm. ( g ) Western blot analysis of Huh-7 cells transfected with the indicated plasmids. EGFP-N1 was co-transfected to monitor transfection efficiency. ( h ) HEK293 cells were transfected with GST-NS5A along with the indicated plasmids for 48 h. Total cell lysates were incubated with glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( i , j ) Huh-7 cells were transfected with the indicated plasmids followed by HCVcc infection (10 MOI) for 3 days before harvesting. The intracellular HCV RNA levels were determined using RT–qPCR ( i ), and total cell lysates were subjected to immunoblotting using the indicated antibodies ( j ). The bars indicate the mean value±s.d. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. ** P value

    Techniques Used: Transfection, Plasmid Preparation, Construct, GST Pulldown Assay, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Microscopy, Western Blot, Incubation, Infection, Quantitative RT-PCR

    38) Product Images from "Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig"

    Article Title: Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158046

    Morphological changes of piPSCs in response to culture conditions. The piPS-14 cells cultured under LIF conditions were transferred to two different media supplemented with LIF + 2i or bFGF. (A) Four days after changing culture media, the cells cultured in bFGF started to change morphologically, becoming flattened. (B) Later, the cells cultured with bFGF showed a primed-like flattened morphology, while those cultured with LIF or LIF + 2i still showed compact dome-shaped morphology. (C) When cultured in the absence of dox, in all cells under the three conditions, the number of colonies gradually decreased within 4 days, and AP-positive colonies were absent after subcultures. Scale bar = 200 μm.
    Figure Legend Snippet: Morphological changes of piPSCs in response to culture conditions. The piPS-14 cells cultured under LIF conditions were transferred to two different media supplemented with LIF + 2i or bFGF. (A) Four days after changing culture media, the cells cultured in bFGF started to change morphologically, becoming flattened. (B) Later, the cells cultured with bFGF showed a primed-like flattened morphology, while those cultured with LIF or LIF + 2i still showed compact dome-shaped morphology. (C) When cultured in the absence of dox, in all cells under the three conditions, the number of colonies gradually decreased within 4 days, and AP-positive colonies were absent after subcultures. Scale bar = 200 μm.

    Techniques Used: Cell Culture

    39) Product Images from "Equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro"

    Article Title: Equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt479

    Percentage inhibition of S. equi superantigen (sAg)-induced peripheral blood mononuclear cells (PBMCs) proliferation when co-cultured with mesenchymal stem cells (MSCs) at ratios of 1:10, 1:66, 1:200, and 1:400 (MSC to PBMC). Error bars represent the standard error of the mean of three biologic repeats. No significant differences are observed between the ratios ( P > 0.05).
    Figure Legend Snippet: Percentage inhibition of S. equi superantigen (sAg)-induced peripheral blood mononuclear cells (PBMCs) proliferation when co-cultured with mesenchymal stem cells (MSCs) at ratios of 1:10, 1:66, 1:200, and 1:400 (MSC to PBMC). Error bars represent the standard error of the mean of three biologic repeats. No significant differences are observed between the ratios ( P > 0.05).

    Techniques Used: Inhibition, Cell Culture

    Proliferation of equine PBMCs is suppressed by co-culture with equine MSCs. (A) Immunocytochemical staining of IFN-γ-treated mesenchymal stem cells (MSCs) for MHC I and MHC II. Cell nuclei are indicated by blue Dapi staining, and expressed MHC I or II proteins, by green staining. Data show representative images from one of three replicates. (B) Relative proliferation of effector (Eff#1) peripheral blood mononuclear cells (PBMCs) to mesenchymal stem cells (MSCs) cultured in the presence and absence of IFN-γ. Autologous and allogeneic stimulator cells are used as negative and positive controls (Stim #1 and Stim #2, respectively). *Results significantly different from those for nonactivated (NA) PBMCs ( P
    Figure Legend Snippet: Proliferation of equine PBMCs is suppressed by co-culture with equine MSCs. (A) Immunocytochemical staining of IFN-γ-treated mesenchymal stem cells (MSCs) for MHC I and MHC II. Cell nuclei are indicated by blue Dapi staining, and expressed MHC I or II proteins, by green staining. Data show representative images from one of three replicates. (B) Relative proliferation of effector (Eff#1) peripheral blood mononuclear cells (PBMCs) to mesenchymal stem cells (MSCs) cultured in the presence and absence of IFN-γ. Autologous and allogeneic stimulator cells are used as negative and positive controls (Stim #1 and Stim #2, respectively). *Results significantly different from those for nonactivated (NA) PBMCs ( P

    Techniques Used: Co-Culture Assay, Staining, Cell Culture

    Concentration of cytokines produced by nonactivated (NA) and S. equi superantigen (sAg)-stimulated peripheral blood mononuclear cells (PBMCs) after exposure to 24-, 48-, and 72-hour MSC-conditioned media, measured with ELISA (that is, NA24 represents nonactivated PBMCs exposed to 24-hour MSC-conditioned media). Error bars represent the standard error of the mean from three individual experiments using three cell lines. *Results significantly different from those for nonactivated PBMCs (NA), ¥, Values significantly different ( P
    Figure Legend Snippet: Concentration of cytokines produced by nonactivated (NA) and S. equi superantigen (sAg)-stimulated peripheral blood mononuclear cells (PBMCs) after exposure to 24-, 48-, and 72-hour MSC-conditioned media, measured with ELISA (that is, NA24 represents nonactivated PBMCs exposed to 24-hour MSC-conditioned media). Error bars represent the standard error of the mean from three individual experiments using three cell lines. *Results significantly different from those for nonactivated PBMCs (NA), ¥, Values significantly different ( P

    Techniques Used: Concentration Assay, Produced, Enzyme-linked Immunosorbent Assay

    Proliferation of peripheral blood mononuclear cells (PBMCs) is increased with either S. equi super-antigens (sAg) or phytohemagglutinin (PHA) compared with nonactivated (NA) PBMCs and is inhibited by co-culture with allogeneic mesenchymal stem cells (MSCs). Graph depicts radioactive thymidine ( 3 H-thymidine) counts per minute (cpm) as a measure of cell proliferation. Error bars represent the standard deviation of triplicate wells of the same experiment, which is representative of the three replicates.
    Figure Legend Snippet: Proliferation of peripheral blood mononuclear cells (PBMCs) is increased with either S. equi super-antigens (sAg) or phytohemagglutinin (PHA) compared with nonactivated (NA) PBMCs and is inhibited by co-culture with allogeneic mesenchymal stem cells (MSCs). Graph depicts radioactive thymidine ( 3 H-thymidine) counts per minute (cpm) as a measure of cell proliferation. Error bars represent the standard deviation of triplicate wells of the same experiment, which is representative of the three replicates.

    Techniques Used: Co-Culture Assay, Standard Deviation

    Soluble factors are involved in MSC-mediated PMBC suppression. (A) Mesenchymal stem cells (MSCs) suppress S. equi superantigen (sAg)-induced peripheral blood mononuclear cells (PBMCs) proliferation when separated by a transwell membrane (TW). Graph depicts radioactive thymidine ( 3 H-thymidine) counts per minute (cpm) as a measure of cell proliferation. Differing letter annotations denote a significantly different mean (ANOVA, all P
    Figure Legend Snippet: Soluble factors are involved in MSC-mediated PMBC suppression. (A) Mesenchymal stem cells (MSCs) suppress S. equi superantigen (sAg)-induced peripheral blood mononuclear cells (PBMCs) proliferation when separated by a transwell membrane (TW). Graph depicts radioactive thymidine ( 3 H-thymidine) counts per minute (cpm) as a measure of cell proliferation. Differing letter annotations denote a significantly different mean (ANOVA, all P

    Techniques Used:

    Proliferation of equine PBMCs is not induced by co-culture with equine ESCs. (A) Immunocytochemical staining of IFN-γ-treated embryo-derived stem cells (ESCs) for MHC I and MHC II. Cell nuclei are indicated by blue Dapi staining, and expressed MHC I or II proteins, by green staining. Representative images from one of three replicates are shown. (B) Relative proliferation of peripheral blood mononuclear cells (PBMCs) to undifferentiated (ESCs) and differentiated (dES) ESCs cultured in the presence and absence of IFN-γ, where NA is baseline, nonactivated PBMC proliferation; sAg is superantigen-stimulated PBMCs (positive control); IFN-γ is 72-hour pretreated undifferentiated or differentiated ESC. *Results significantly different relative proliferation when compared with NA PBMCs ( P
    Figure Legend Snippet: Proliferation of equine PBMCs is not induced by co-culture with equine ESCs. (A) Immunocytochemical staining of IFN-γ-treated embryo-derived stem cells (ESCs) for MHC I and MHC II. Cell nuclei are indicated by blue Dapi staining, and expressed MHC I or II proteins, by green staining. Representative images from one of three replicates are shown. (B) Relative proliferation of peripheral blood mononuclear cells (PBMCs) to undifferentiated (ESCs) and differentiated (dES) ESCs cultured in the presence and absence of IFN-γ, where NA is baseline, nonactivated PBMC proliferation; sAg is superantigen-stimulated PBMCs (positive control); IFN-γ is 72-hour pretreated undifferentiated or differentiated ESC. *Results significantly different relative proliferation when compared with NA PBMCs ( P

    Techniques Used: Co-Culture Assay, Staining, Derivative Assay, Cell Culture, Positive Control

    mRNA expression by nonactivated (NA) and S. equi superantigens (sAg)-stimulated peripheral blood mononuclear cells (PBMCs) after exposure to 24-, 48-, and 72-hour MSC conditioned media (that is, NA24 represents nonactivated PBMCs exposed to 24-hour MSC-conditioned media). Results represented as change in fold expression relative to NA PBMCs (control conditions), adjusted to the housekeeping gene 18S. Error bars represent the standard error of the mean from three individual experiments using three cell lines. *Results significantly different ( P
    Figure Legend Snippet: mRNA expression by nonactivated (NA) and S. equi superantigens (sAg)-stimulated peripheral blood mononuclear cells (PBMCs) after exposure to 24-, 48-, and 72-hour MSC conditioned media (that is, NA24 represents nonactivated PBMCs exposed to 24-hour MSC-conditioned media). Results represented as change in fold expression relative to NA PBMCs (control conditions), adjusted to the housekeeping gene 18S. Error bars represent the standard error of the mean from three individual experiments using three cell lines. *Results significantly different ( P

    Techniques Used: Expressing

    40) Product Images from "MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A"

    Article Title: MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A

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

    doi:

    ( A ) Expression of poliovirus 3A from rVV-3A,GFP. Immunoblot analysis of lysates from chimpanzee lymphocytes that were mock-infected, infected with rVV-3A/GFP, or coinfected with rVV-3A/GFP and rVV-E2/NS3. Time courses in hours after infection (h.p.i.) are shown. Lysate from poliovirus-infected HeLa cells shows both 3A and its precursor 3AB. ( B ) Effect of 3A protein on cell-surface expression of newly synthesized MHC class I. Chimpanzee B lymphoblastoid cells were infected with rVV-GFP or rVV-3A,GFP, labeled with [ 35 S]methionine and [ 35 S]cysteine for 15 min, and subsequently incubated with nonradioactive amino acids for various periods of time. Molecules on the cell surface were subjected to biotinylation and recovered by use of streptavidin agarose. Total (T) MHC class I molecules after 60 min of chase and MHC class I molecules that appeared at the cell surface after the indicated times (min) were isolated by immunoprecipitation with anti-MHC class I antibody w6/32. Molecular masses (kDa) of marker proteins are indicated. ( C ) Effect of poliovirus 3A on target-cell lysis by CTL. Chimpanzee B lymphoblastoid target cells were infected with wild-type vaccinia (○), infected with rVV-E2/NS3 alone (■), or coinfected with rVV-E2/NS3 and rVV-GFP (●) or rVV-E2/NS3 and rVV-3A,GFP (▴). At 12 h postinfection, target cells were labeled with 51 Cr and incubated for 4 h with T91 CTL at the indicated effector/target ratios. Specific lysis was expressed as the amount of 51 Cr released into the supernatant as a percentage of the total counts per sample. Data are presented as the mean of triplicate samples, and the standard deviations are indicated.
    Figure Legend Snippet: ( A ) Expression of poliovirus 3A from rVV-3A,GFP. Immunoblot analysis of lysates from chimpanzee lymphocytes that were mock-infected, infected with rVV-3A/GFP, or coinfected with rVV-3A/GFP and rVV-E2/NS3. Time courses in hours after infection (h.p.i.) are shown. Lysate from poliovirus-infected HeLa cells shows both 3A and its precursor 3AB. ( B ) Effect of 3A protein on cell-surface expression of newly synthesized MHC class I. Chimpanzee B lymphoblastoid cells were infected with rVV-GFP or rVV-3A,GFP, labeled with [ 35 S]methionine and [ 35 S]cysteine for 15 min, and subsequently incubated with nonradioactive amino acids for various periods of time. Molecules on the cell surface were subjected to biotinylation and recovered by use of streptavidin agarose. Total (T) MHC class I molecules after 60 min of chase and MHC class I molecules that appeared at the cell surface after the indicated times (min) were isolated by immunoprecipitation with anti-MHC class I antibody w6/32. Molecular masses (kDa) of marker proteins are indicated. ( C ) Effect of poliovirus 3A on target-cell lysis by CTL. Chimpanzee B lymphoblastoid target cells were infected with wild-type vaccinia (○), infected with rVV-E2/NS3 alone (■), or coinfected with rVV-E2/NS3 and rVV-GFP (●) or rVV-E2/NS3 and rVV-3A,GFP (▴). At 12 h postinfection, target cells were labeled with 51 Cr and incubated for 4 h with T91 CTL at the indicated effector/target ratios. Specific lysis was expressed as the amount of 51 Cr released into the supernatant as a percentage of the total counts per sample. Data are presented as the mean of triplicate samples, and the standard deviations are indicated.

    Techniques Used: Expressing, Infection, Synthesized, Labeling, Incubation, Isolation, Immunoprecipitation, Marker, Lysis, CTL Assay

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    Transfection:

    Article Title: Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes
    Article Snippet: .. For western blot experiments, Neuro-2a cells transfected with siRNA targeting myosin II isoforms, IIA1, IIA2, IIB1 or IIB2, were washed with ice-cold PBS and scraped in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1.2% Triton X-100, 0.1% 2-mercaptoethanol, 0.5 mM EDTA, and protease inhibitors (Sigma) for 20 min. ..

    Mutagenesis:

    Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2
    Article Snippet: .. Wild-type and FANCD2 mutant DT40 cell lines are described in and were cultured in RPMI medium supplemented with 10% FCS, 1% chicken serum (Sigma Aldrich) and 10 μM β-mercaptoethanol (Life Technologies). ..

    Cell Culture:

    Article Title: Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation
    Article Snippet: .. EPC culture with E2 β or P4 To assess the effects of E2 β or P4 on EPC proliferation of EPCs, 8 × 105 PBMCs, derived from the peripheral blood of women in the menstrual or luteal phases, were seeded into each well of 96-well culture plates coated with human fibronectin (Sigma-Aldrich), and cultured in endothelial basal medium (phenol red free EBM-2, Clonetics; San Diego, CA) supplemented with EGM-2MV (Clonetics) consisting of 5% charcoal stripped serum (Invitrogen), VEGF, FGF2, EGF, IGF1, and ascorbic acid with or without 10-9 -10-7 M of E2 β or P4 . .. ICI 182,780 (10-5 M, Wako Pure Chemical Industries, Ltd., Osaka, Japan) or RU486 (10-5 M, Sigma-Aldrich) was used as an inhibitor of ER or PR, respectively [ ].

    Article Title: Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells
    Article Snippet: .. Culture of mouse ESCs Nkx2.5-GFP/SHF-dsRed (RG) mouse ESCs [ ] were cultured in knockout Dulbecco's Modified Eagle Medium (DMEM, Sigma) with 15% Fetal Bovine Serum (FBS, Hyclone, Utah, US), 1% penicillin/streptomycin solution (Life Technologies), 2 mM L-glutamine (Life Technologies), 1% non-essential aminoacids (Life Technologies), 0.1 mM-mercaptoethanol (Sigma) and 1000 U/mL leukemia inhibitory factor (LIF; Chemicon, Temecula, Ca, USA). .. Mouse ESCs were cultured in 0.1% gelatin coated dishes at 37°C/5%CO2 .

    Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2
    Article Snippet: .. Wild-type and FANCD2 mutant DT40 cell lines are described in and were cultured in RPMI medium supplemented with 10% FCS, 1% chicken serum (Sigma Aldrich) and 10 μM β-mercaptoethanol (Life Technologies). ..

    Article Title: Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors
    Article Snippet: .. Cell Culture iPS and iTP cells (induced cells described above) were maintained on an MEF feeder layer in DMEM-F12 (Sigma-Aldrich), 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1:100 dilution of nonessential amino acids (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 5 ng/mL bFGF (Repro CELL), and penicillin/streptomycin (Sigma-Aldrich). .. For passaging, iPS/iTP colonies were dissociated with Dissociation Solution for human ESCs/iPSCs (Riken CDB, Kobe, Japan) and split at ratios between 1:3 and 1:6.

    Labeling:

    Article Title: CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans
    Article Snippet: .. Co-localization of GDT1-GFP protein and Golgi-tracker red dye For Golgi apparatus labeling, the pmr1/pmr1 GDT1-GFP/gdt1 cells were grown to log-phase in SD-URA, harvested, washed twice with PBS, and suspended in 200 μl PBS supplemented with 4 μl beta-mercaptoethanol and 15 U lyticase enzyme (Sigma). .. Cells was incubated at 37 °C for 40 min to partially digest the cell wall, and were washed once with PBS and suspended in 200 μl PBS before they were mixed with 2 μl Golgi-Tracker Red dye (33.3 mg ml− 1 ) (Beyotime Institute of Biochemistry, China) and incubated at 4 °C for 30 min.

    Purification:

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    other:

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    Article Snippet: Drugs (±)-trans-U-50488H (U50,488 H), [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) [D-Pen2,5,P-CL-Phe4]Enkephalin (DPDPE), nor-binaltorphimine (nor-BNI), β-funaltrexamine (β-FNA), naloxone, GTPγS and GDP were obtained from Sigma-Aldrich (St. Louis, MO).

    Knock-Out:

    Article Title: Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells
    Article Snippet: .. Culture of mouse ESCs Nkx2.5-GFP/SHF-dsRed (RG) mouse ESCs [ ] were cultured in knockout Dulbecco's Modified Eagle Medium (DMEM, Sigma) with 15% Fetal Bovine Serum (FBS, Hyclone, Utah, US), 1% penicillin/streptomycin solution (Life Technologies), 2 mM L-glutamine (Life Technologies), 1% non-essential aminoacids (Life Technologies), 0.1 mM-mercaptoethanol (Sigma) and 1000 U/mL leukemia inhibitory factor (LIF; Chemicon, Temecula, Ca, USA). .. Mouse ESCs were cultured in 0.1% gelatin coated dishes at 37°C/5%CO2 .

    Western Blot:

    Article Title: Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes
    Article Snippet: .. For western blot experiments, Neuro-2a cells transfected with siRNA targeting myosin II isoforms, IIA1, IIA2, IIB1 or IIB2, were washed with ice-cold PBS and scraped in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1.2% Triton X-100, 0.1% 2-mercaptoethanol, 0.5 mM EDTA, and protease inhibitors (Sigma) for 20 min. ..

    Modification:

    Article Title: Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells
    Article Snippet: .. Culture of mouse ESCs Nkx2.5-GFP/SHF-dsRed (RG) mouse ESCs [ ] were cultured in knockout Dulbecco's Modified Eagle Medium (DMEM, Sigma) with 15% Fetal Bovine Serum (FBS, Hyclone, Utah, US), 1% penicillin/streptomycin solution (Life Technologies), 2 mM L-glutamine (Life Technologies), 1% non-essential aminoacids (Life Technologies), 0.1 mM-mercaptoethanol (Sigma) and 1000 U/mL leukemia inhibitory factor (LIF; Chemicon, Temecula, Ca, USA). .. Mouse ESCs were cultured in 0.1% gelatin coated dishes at 37°C/5%CO2 .

    Lysis:

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    Derivative Assay:

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  • 99
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    Kinetic analysis of editing of HNV by ThrRS at 37 °C and pH 8. All reactions were performed using HNV as the substrate. Panel (A), linear rate of AMP formation in the presence (open circles) and absence (filled circles) of tRNA Thr . Panel (B), enzyme-independent hydrolysis of HNV-AMP in Buffer A. Panels (C) and (D), rate of deacylation of HNV-[ 32 P] tRNA Thr  in the presence and absence of wild type ThrRS, respectively.
    Buffer A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
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    LCD shows direct differentiation outcomes of <t>H9</t> cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with <t>KSR</t> and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P
    Ksr Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Depletion of kinesin-5 reduces the distance of axonal retraction. (A and B) DIC images of control axons (A) and kinesin-5–depleted axons (B) before and 30 min after treatment with 0.3 mM <t>noc-7.</t> (B) Kinesin-5–depleted axons continue to elongate even in the presence of noc-7. Arrows demarcate the distal tips of growth cones before and after noc-7 treatment. (C) Quantitative analysis of noc-7–induced axonal retraction revealed a statistically significant reduction in mean distance retracted in neurons depleted of kinesin-5 (mean ± SEM; control, n = 54; kinesin-5 siRNA, n = 55; *, P = 0.011). Bar, 20 μm.
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    Image Search Results


    Kinetic analysis of editing of HNV by ThrRS at 37 °C and pH 8. All reactions were performed using HNV as the substrate. Panel (A), linear rate of AMP formation in the presence (open circles) and absence (filled circles) of tRNA Thr . Panel (B), enzyme-independent hydrolysis of HNV-AMP in Buffer A. Panels (C) and (D), rate of deacylation of HNV-[ 32 P] tRNA Thr  in the presence and absence of wild type ThrRS, respectively.

    Journal: Biochemistry

    Article Title: Fidelity escape by the unnatural amino acid ?-hydroxynorvaline: an efficient substrate for Escherichia coli threonyl-tRNA synthetase with toxic effects on growth †

    doi: 10.1021/bi101360a

    Figure Lengend Snippet: Kinetic analysis of editing of HNV by ThrRS at 37 °C and pH 8. All reactions were performed using HNV as the substrate. Panel (A), linear rate of AMP formation in the presence (open circles) and absence (filled circles) of tRNA Thr . Panel (B), enzyme-independent hydrolysis of HNV-AMP in Buffer A. Panels (C) and (D), rate of deacylation of HNV-[ 32 P] tRNA Thr in the presence and absence of wild type ThrRS, respectively.

    Article Snippet: For both proteins, the final fractions were pooled, dialyzed against Buffer A (10 mM Tris pH 8.0, 100 mM KCl, 10 mM MgCl2 , 3 mM β-mercaptoethanol), and then concentrated under low speed centrifugation using Millipore concentrators, prior to storage in 50% glycerol at −20 °C.

    Techniques:

    LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Journal: Biochemical and biophysical research communications

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    doi: 10.1016/j.bbrc.2014.08.137

    Figure Lengend Snippet: LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Article Snippet: Briefly, H9 cells were cultured on MEFs in KSR medium (DMEM/F12, 20 % KSR, 0.1 mM β-mercaptoethanol, 10 ng/ml of FGF-2) and disaggregated using accutase (Millipore, Billerica, MA, USA) for 20 min, washed with KSR medium and pre-plated on gelatin-coated 6-well plates for 1 h at 37 °C in the presence of the ROCK inhibitor (Y-27632) to remove MEFs.

    Techniques: Derivative Assay, Software

    Synergistic contribution of SMAD signaling blockers and localized high cell density in NE differentiation A-F, Five days after the cell-clump-based differentiation of NE in KSR and N2 medium with (D-F) or without (A-C) SMAD signaling blockers, H9-derived cells were subjected to the IF assay using anti-PAX6 antibody (green, A-D). The nuclei were stained using DAPI (blue, C and D). The micrographs were divided into 20 (5×4) squares as indicated (C and D). E-F , The number of total cells and PAX6-positive cells in each square was quantified using Image J software. The ratio of PAX6-positive cells to total cells in each square was determined. The squares with equivalent ratios were binned together. The ratios of PAX6-positive cells to total cells in each bin are shown (F, X±SD, **P

    Journal: Biochemical and biophysical research communications

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    doi: 10.1016/j.bbrc.2014.08.137

    Figure Lengend Snippet: Synergistic contribution of SMAD signaling blockers and localized high cell density in NE differentiation A-F, Five days after the cell-clump-based differentiation of NE in KSR and N2 medium with (D-F) or without (A-C) SMAD signaling blockers, H9-derived cells were subjected to the IF assay using anti-PAX6 antibody (green, A-D). The nuclei were stained using DAPI (blue, C and D). The micrographs were divided into 20 (5×4) squares as indicated (C and D). E-F , The number of total cells and PAX6-positive cells in each square was quantified using Image J software. The ratio of PAX6-positive cells to total cells in each square was determined. The squares with equivalent ratios were binned together. The ratios of PAX6-positive cells to total cells in each bin are shown (F, X±SD, **P

    Article Snippet: Briefly, H9 cells were cultured on MEFs in KSR medium (DMEM/F12, 20 % KSR, 0.1 mM β-mercaptoethanol, 10 ng/ml of FGF-2) and disaggregated using accutase (Millipore, Billerica, MA, USA) for 20 min, washed with KSR medium and pre-plated on gelatin-coated 6-well plates for 1 h at 37 °C in the presence of the ROCK inhibitor (Y-27632) to remove MEFs.

    Techniques: Derivative Assay, Staining, Software

    Depletion of kinesin-5 reduces the distance of axonal retraction. (A and B) DIC images of control axons (A) and kinesin-5–depleted axons (B) before and 30 min after treatment with 0.3 mM noc-7. (B) Kinesin-5–depleted axons continue to elongate even in the presence of noc-7. Arrows demarcate the distal tips of growth cones before and after noc-7 treatment. (C) Quantitative analysis of noc-7–induced axonal retraction revealed a statistically significant reduction in mean distance retracted in neurons depleted of kinesin-5 (mean ± SEM; control, n = 54; kinesin-5 siRNA, n = 55; *, P = 0.011). Bar, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: Kinesin-5 regulates the growth of the axon by acting as a brake on its microtubule array

    doi: 10.1083/jcb.200702074

    Figure Lengend Snippet: Depletion of kinesin-5 reduces the distance of axonal retraction. (A and B) DIC images of control axons (A) and kinesin-5–depleted axons (B) before and 30 min after treatment with 0.3 mM noc-7. (B) Kinesin-5–depleted axons continue to elongate even in the presence of noc-7. Arrows demarcate the distal tips of growth cones before and after noc-7 treatment. (C) Quantitative analysis of noc-7–induced axonal retraction revealed a statistically significant reduction in mean distance retracted in neurons depleted of kinesin-5 (mean ± SEM; control, n = 54; kinesin-5 siRNA, n = 55; *, P = 0.011). Bar, 20 μm.

    Article Snippet: DIC images of axons were recorded before and 30 min after addition of noc-7.

    Techniques: