s2 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher s2 cells
    Knockdown of Dm mtSSB endo and overexpression of Dm mtSSB variants in <t>S2</t> cells causes mtDNA depletion under conditions of mitochondrial homeostasis. A , cells carrying pMt/ Dm mtSSBwt/Hy were cultured in the presence of dsRNA homologous to the 3′-UTR
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    Images

    1) Product Images from "Reduced Stimulation of Recombinant DNA Polymerase ? and Mitochondrial DNA (mtDNA) Helicase by Variants of Mitochondrial Single-stranded DNA-binding Protein (mtSSB) Correlates with Defects in mtDNA Replication in Animal Cells *"

    Article Title: Reduced Stimulation of Recombinant DNA Polymerase ? and Mitochondrial DNA (mtDNA) Helicase by Variants of Mitochondrial Single-stranded DNA-binding Protein (mtSSB) Correlates with Defects in mtDNA Replication in Animal Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.289983

    Knockdown of Dm mtSSB endo and overexpression of Dm mtSSB variants in S2 cells causes mtDNA depletion under conditions of mitochondrial homeostasis. A , cells carrying pMt/ Dm mtSSBwt/Hy were cultured in the presence of dsRNA homologous to the 3′-UTR
    Figure Legend Snippet: Knockdown of Dm mtSSB endo and overexpression of Dm mtSSB variants in S2 cells causes mtDNA depletion under conditions of mitochondrial homeostasis. A , cells carrying pMt/ Dm mtSSBwt/Hy were cultured in the presence of dsRNA homologous to the 3′-UTR

    Techniques Used: Over Expression, Cell Culture

    Expression of  Dm mtSSB variants impedes recovery from mtDNA depletion in  Drosophila  S2 cells. A ,  left panel , 0.2 μg/ml of EtBr was applied for 3 days to the growth media of cells carrying the  Dm mtSSBwt gene, followed by a recovery time of 12 days
    Figure Legend Snippet: Expression of Dm mtSSB variants impedes recovery from mtDNA depletion in Drosophila S2 cells. A , left panel , 0.2 μg/ml of EtBr was applied for 3 days to the growth media of cells carrying the Dm mtSSBwt gene, followed by a recovery time of 12 days

    Techniques Used: Expressing

    2) Product Images from "Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator"

    Article Title: Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator

    Journal: Biochemical Journal

    doi: 10.1042/BJ20041078

    Western blot analysis of recombinant anosmin-1 in Drosophila S2 cells ( a ) Transient expression of recombinant anosmin-1 was induced by 500 μM CuSO 4 in 10% serum-containing medium. Recombinant anosmin-1 proteins present in cell lysate (L), salt extract (S) and conditioned medium (M) were identified using an anti-His monoclonal antibody. The molecular-mass-markers are shown. Transiently transfected Drosophila S2 cells in serum-free medium culture were induced to express proteins in the absence ( b ) and presence ( c ) of 100 μg/ml HS. ( d ) PIWF1 and PIWF4 proteins generated from stable Drosophila S2 cell line in serum-free medium were analysed in presence (+) or absence (−) of HS in comparison with vector (control), GFP, mPIWF1 172 and mPIWF4 172 .
    Figure Legend Snippet: Western blot analysis of recombinant anosmin-1 in Drosophila S2 cells ( a ) Transient expression of recombinant anosmin-1 was induced by 500 μM CuSO 4 in 10% serum-containing medium. Recombinant anosmin-1 proteins present in cell lysate (L), salt extract (S) and conditioned medium (M) were identified using an anti-His monoclonal antibody. The molecular-mass-markers are shown. Transiently transfected Drosophila S2 cells in serum-free medium culture were induced to express proteins in the absence ( b ) and presence ( c ) of 100 μg/ml HS. ( d ) PIWF1 and PIWF4 proteins generated from stable Drosophila S2 cell line in serum-free medium were analysed in presence (+) or absence (−) of HS in comparison with vector (control), GFP, mPIWF1 172 and mPIWF4 172 .

    Techniques Used: Western Blot, Recombinant, Expressing, Transfection, Generated, Plasmid Preparation

    3) Product Images from "Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes"

    Article Title: Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200612011

    Dm-Kat60 stimulates Pacman, whereas Dm-Spastin and Dm-Fidgetin drive poleward flux. (A) Frames of time-lapse recordings of anaphase EGFP–α-tubulin expressing S2 cells in which chromosomes were Hoechst stained just before visualization, and bars were photobleached across each half spindle. Yellow arrowheads mark the initial position of bleach marks; yellow arrows and dots mark their current positions. Blue arrowheads mark the initial positions of the leading edge of a selected chromosome; blue arrows and dots mark the current positions of the chromosome. The control spindle displays both poleward flux and Pacman motility (manifested as chromosomes overtaking bleach marks), but the Dm-Kat60 RNAi spindle has visibly diminished Pacman motility, and the Dm-Spastin and Dm-Fidgetin spindles have diminished flux. (B) Representative plots of distance versus time of individual chromosomes measured from the RNAi-treated cells in A. Red lines represent flux, and blue lines represent Pacman motility (measured as the approach of chromosomes to bleach marks). (C) Mean flux, Pacman, and chromatid-to-pole velocities (+SD) for anaphase spindles. Statistical differences from the control are marked with asterisks. Numbers in parentheses are sample sizes.
    Figure Legend Snippet: Dm-Kat60 stimulates Pacman, whereas Dm-Spastin and Dm-Fidgetin drive poleward flux. (A) Frames of time-lapse recordings of anaphase EGFP–α-tubulin expressing S2 cells in which chromosomes were Hoechst stained just before visualization, and bars were photobleached across each half spindle. Yellow arrowheads mark the initial position of bleach marks; yellow arrows and dots mark their current positions. Blue arrowheads mark the initial positions of the leading edge of a selected chromosome; blue arrows and dots mark the current positions of the chromosome. The control spindle displays both poleward flux and Pacman motility (manifested as chromosomes overtaking bleach marks), but the Dm-Kat60 RNAi spindle has visibly diminished Pacman motility, and the Dm-Spastin and Dm-Fidgetin spindles have diminished flux. (B) Representative plots of distance versus time of individual chromosomes measured from the RNAi-treated cells in A. Red lines represent flux, and blue lines represent Pacman motility (measured as the approach of chromosomes to bleach marks). (C) Mean flux, Pacman, and chromatid-to-pole velocities (+SD) for anaphase spindles. Statistical differences from the control are marked with asterisks. Numbers in parentheses are sample sizes.

    Techniques Used: Expressing, Staining

    Dm-Kat60, Dm-Spastin, and Dm-Fidgetin are all required for proper chromosome segregation during anaphase A. (A) After RNAi, EGFP–α-tubulin expressing S2 cells were recorded from metaphase until late anaphase as single confocal sections. Chromosomes are visible as black dots at the ends of the prominent kinetochore fibers. In the last frames, yellow dots mark the positions of the chromosomes, and blue dots the centrosome position, in a half spindle. Numbers are elapsed time after the first frame. (B) Anaphase chromatid-to-pole velocities (+SD) after RNAi. *, P
    Figure Legend Snippet: Dm-Kat60, Dm-Spastin, and Dm-Fidgetin are all required for proper chromosome segregation during anaphase A. (A) After RNAi, EGFP–α-tubulin expressing S2 cells were recorded from metaphase until late anaphase as single confocal sections. Chromosomes are visible as black dots at the ends of the prominent kinetochore fibers. In the last frames, yellow dots mark the positions of the chromosomes, and blue dots the centrosome position, in a half spindle. Numbers are elapsed time after the first frame. (B) Anaphase chromatid-to-pole velocities (+SD) after RNAi. *, P

    Techniques Used: Expressing

    Dm-Spastin and Dm-Fidgetin stimulate the turnover of α-tubulin at MT ends and γ-tubulin at centrosomes. (A, top) FRAP of metaphase spindles of EGFP–α-tubulin expressing S2 cells after RNAi. One spindle pole region (MT minus ends) and one spindle equator region (MT plus ends) were photobleached, and their fluorescence recoveries were recorded. Images are frames from time-lapse recordings; the elapsed times after photobleaching are shown to the left. Fluorescent α-tubulin recovery after Dm-Kat60 RNAi is similar to control, whereas the recoveries after Dm-Spastin and Dm-Fidgetin RNAi are visibly slower. (middle) Mean fluorescence recovery half-times ( t 1/2 ) for MT minus ends (left). Asterisks indicate treatments with statistically slower α-tubulin turnover rates compared with control. Percentage of fluorescence recoveries are shown (right). Numbers within bars are sample sizes, and error bars are SD. (bottom) Mean t 1/2 (+SD) of MT plus ends. (B, top) The two intensely fluorescent centrosomes of γ-tubulin–EGFP expressing S2 cells were photobleached, and their fluorescent recoveries were recorded. Selected time-point images and their postbleach elapsed times are shown. (middle) Mean t 1/2 (+SD) of γ-tubulin turnover at centrosomes. (bottom) Percentage of fluorescence recoveries is shown.
    Figure Legend Snippet: Dm-Spastin and Dm-Fidgetin stimulate the turnover of α-tubulin at MT ends and γ-tubulin at centrosomes. (A, top) FRAP of metaphase spindles of EGFP–α-tubulin expressing S2 cells after RNAi. One spindle pole region (MT minus ends) and one spindle equator region (MT plus ends) were photobleached, and their fluorescence recoveries were recorded. Images are frames from time-lapse recordings; the elapsed times after photobleaching are shown to the left. Fluorescent α-tubulin recovery after Dm-Kat60 RNAi is similar to control, whereas the recoveries after Dm-Spastin and Dm-Fidgetin RNAi are visibly slower. (middle) Mean fluorescence recovery half-times ( t 1/2 ) for MT minus ends (left). Asterisks indicate treatments with statistically slower α-tubulin turnover rates compared with control. Percentage of fluorescence recoveries are shown (right). Numbers within bars are sample sizes, and error bars are SD. (bottom) Mean t 1/2 (+SD) of MT plus ends. (B, top) The two intensely fluorescent centrosomes of γ-tubulin–EGFP expressing S2 cells were photobleached, and their fluorescent recoveries were recorded. Selected time-point images and their postbleach elapsed times are shown. (middle) Mean t 1/2 (+SD) of γ-tubulin turnover at centrosomes. (bottom) Percentage of fluorescence recoveries is shown.

    Techniques Used: Expressing, Fluorescence

    Dm-Spastin and Dm-Fidgetin, but not Dm-Kat60, stimulate poleward flux in metaphase spindles.  (A) To visualize poleward flux, bars were photobleached across metaphase spindles of EGFP–α-tubulin expressing S2 cells, after RNAi treatment. Images are frames taken from time-lapse videos; the elapsed times (s) after photobleaching are indicated to the left or within the panels. Orange arrowheads mark the initial position of the photobleached bars, and blue arrowheads indicate their current positions. Kymographs were generated from the time-lapse recordings of these spindles to further illustrate the flux rates: a large angle between the tracks of bleached bar and pole indicates a relatively high flux rate (control and Dm-Kat60 RNAi), whereas more parallel tracks indicate decreased flux (Dm-Spastin and Dm-Fidgetin RNAi). t and d indicate the time and distance axes, respectively. (B) Mean flux rates (+SD) for metaphase cells after RNAi treatment. The flux rate after Dm-Kat60 RNAi is equivalent to control, whereas flux is significantly decreased after Dm-Spastin or Dm-Fidgetin RNAi. *, P
    Figure Legend Snippet: Dm-Spastin and Dm-Fidgetin, but not Dm-Kat60, stimulate poleward flux in metaphase spindles. (A) To visualize poleward flux, bars were photobleached across metaphase spindles of EGFP–α-tubulin expressing S2 cells, after RNAi treatment. Images are frames taken from time-lapse videos; the elapsed times (s) after photobleaching are indicated to the left or within the panels. Orange arrowheads mark the initial position of the photobleached bars, and blue arrowheads indicate their current positions. Kymographs were generated from the time-lapse recordings of these spindles to further illustrate the flux rates: a large angle between the tracks of bleached bar and pole indicates a relatively high flux rate (control and Dm-Kat60 RNAi), whereas more parallel tracks indicate decreased flux (Dm-Spastin and Dm-Fidgetin RNAi). t and d indicate the time and distance axes, respectively. (B) Mean flux rates (+SD) for metaphase cells after RNAi treatment. The flux rate after Dm-Kat60 RNAi is equivalent to control, whereas flux is significantly decreased after Dm-Spastin or Dm-Fidgetin RNAi. *, P

    Techniques Used: Expressing, Generated

    Overexpression of Dm-Kat60, Dm-Spastin, or Dm-Fidgetin eliminates MTs in interphase S2 cells. (A–C) mRFP–α-tubulin expressing S2 cells were transiently transfected with full-length AAA-EGFP constructs. AAA refers specifically to Dm-Kat60, Dm-Spastin, and Dm-Fidgetin. Fluorescent MTs are pseudocolored green; expressed AAA fusion protein is pseudocolored red. Overexpression of any one of the AAA constructs visibly decreases the MT polymer mass. Bars, 5 μm. (D) Mean fluorescence intensities (+SD) of mRFP– α-tubulin for live whole cells after transient transfection with the indicated full-length AAA-EGFP construct. Red bars are tubulin fluorescence intensities of cells that visibly expressed fluorescent AAA protein. Gray bars are intensities of neighboring cells not visibly expressing fluorescent AAA protein. *, P
    Figure Legend Snippet: Overexpression of Dm-Kat60, Dm-Spastin, or Dm-Fidgetin eliminates MTs in interphase S2 cells. (A–C) mRFP–α-tubulin expressing S2 cells were transiently transfected with full-length AAA-EGFP constructs. AAA refers specifically to Dm-Kat60, Dm-Spastin, and Dm-Fidgetin. Fluorescent MTs are pseudocolored green; expressed AAA fusion protein is pseudocolored red. Overexpression of any one of the AAA constructs visibly decreases the MT polymer mass. Bars, 5 μm. (D) Mean fluorescence intensities (+SD) of mRFP– α-tubulin for live whole cells after transient transfection with the indicated full-length AAA-EGFP construct. Red bars are tubulin fluorescence intensities of cells that visibly expressed fluorescent AAA protein. Gray bars are intensities of neighboring cells not visibly expressing fluorescent AAA protein. *, P

    Techniques Used: Over Expression, Expressing, Transfection, Construct, Fluorescence

    4) Product Images from "RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway"

    Article Title: RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201006131

    S2 cell screen identifies genes that regulate the surface expression of dVMAT. (A) The flow chart illustrates the procedure used for screening. S2 cells transfected with GFP-/HA-dVMAT were treated twice with dsRNA over a 6-d period in a 96-well plate, incubated with external HA antibody conjugated to Alexa Fluor 647 for 2 h, washed, and the fluorescence of both GFP and Alexa Fluor 647 was measured at the level of individual cells by flow cytometry. Of 7,200 genes conserved from Drosophila to mammals, 18 positives (z score ≥ 3) were identified and retested using nonoverlapping dsRNA. Kolmogorov-Smirnov analysis of the cumulative frequency distribution reveals that 16 of 18 genes were again positive (P
    Figure Legend Snippet: S2 cell screen identifies genes that regulate the surface expression of dVMAT. (A) The flow chart illustrates the procedure used for screening. S2 cells transfected with GFP-/HA-dVMAT were treated twice with dsRNA over a 6-d period in a 96-well plate, incubated with external HA antibody conjugated to Alexa Fluor 647 for 2 h, washed, and the fluorescence of both GFP and Alexa Fluor 647 was measured at the level of individual cells by flow cytometry. Of 7,200 genes conserved from Drosophila to mammals, 18 positives (z score ≥ 3) were identified and retested using nonoverlapping dsRNA. Kolmogorov-Smirnov analysis of the cumulative frequency distribution reveals that 16 of 18 genes were again positive (P

    Techniques Used: Expressing, Flow Cytometry, Transfection, Incubation, Fluorescence, Cytometry

    Classification of genes identified in the screen by mechanism and effect on the regulated secretion of soluble cargo. S2 cells transfected with DE/AA GFP-/HA-dVMAT were treated with dsRNA-targeting genes identified in the screen, and the uptake of external HA antibody was measured as described in Fig. 1 . (left) Representative cumulative frequency distributions are shown for selected genes in each class. Class I genes increase, class II genes decrease, and class III genes have no effect on antibody uptake by DE/AA dVMAT. (middle) S2 cells expressing ANF-GFP were treated with dsRNA and basal (unstimulated) secretion of GFP fluorescence determined as in Fig. 1 C . Secretion was normalized to cellular ANF-GFP and expressed as a percentage of the fluorescence secreted by control cells. (right) S2 cells expressing ANF-GFP were treated with dsRNA and LPS-induced secretion measured as in Fig. 1 D . *, P
    Figure Legend Snippet: Classification of genes identified in the screen by mechanism and effect on the regulated secretion of soluble cargo. S2 cells transfected with DE/AA GFP-/HA-dVMAT were treated with dsRNA-targeting genes identified in the screen, and the uptake of external HA antibody was measured as described in Fig. 1 . (left) Representative cumulative frequency distributions are shown for selected genes in each class. Class I genes increase, class II genes decrease, and class III genes have no effect on antibody uptake by DE/AA dVMAT. (middle) S2 cells expressing ANF-GFP were treated with dsRNA and basal (unstimulated) secretion of GFP fluorescence determined as in Fig. 1 C . Secretion was normalized to cellular ANF-GFP and expressed as a percentage of the fluorescence secreted by control cells. (right) S2 cells expressing ANF-GFP were treated with dsRNA and LPS-induced secretion measured as in Fig. 1 D . *, P

    Techniques Used: Transfection, Expressing, Fluorescence

    S2 cells express an RSP. (A and B) S2 cells were transiently transfected with wt (black) or DE/AA (red) GFP- and HA-tagged dVMAT, incubated for 2 h at room temperature with external HA antibody conjugated to Alexa Fluor 647, washed, and the fluorescence of individual cells was determined by flow cytometry (A). (inset) The bar graph displays the mean ratio of surface/total fluorescence ( n > 1,800 cells). Kolmogorov-Smirnov analysis of the cumulative frequency distributions binned by surface/total dVMAT ratios (B) indicates a significant change in the DE/AA distribution relative to wt (***, P
    Figure Legend Snippet: S2 cells express an RSP. (A and B) S2 cells were transiently transfected with wt (black) or DE/AA (red) GFP- and HA-tagged dVMAT, incubated for 2 h at room temperature with external HA antibody conjugated to Alexa Fluor 647, washed, and the fluorescence of individual cells was determined by flow cytometry (A). (inset) The bar graph displays the mean ratio of surface/total fluorescence ( n > 1,800 cells). Kolmogorov-Smirnov analysis of the cumulative frequency distributions binned by surface/total dVMAT ratios (B) indicates a significant change in the DE/AA distribution relative to wt (***, P

    Techniques Used: Transfection, Incubation, Fluorescence, Flow Cytometry, Cytometry

    5) Product Images from "Enhancer Sequences Influence the Role of the Amino-Terminal Domain of Bicoid in Transcription"

    Article Title: Enhancer Sequences Influence the Role of the Amino-Terminal Domain of Bicoid in Transcription

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.13.4439-4448.2003

    The amino-terminal domain of Bcd plays different roles on hb and kni enhancer elements. (A) Schematic diagrams of the 250-bp hb enhancer element and the 64-bp kni enhancer element. The Bcd binding sites are represented by arrows. (B) CAT assay results of S2 cells transfected with hb-CAT and kni-CAT reporter plasmids (1 μg) and increasing amounts of an effector plasmid expressing Bcd. Activities obtained with 1 μg of transfected effector plasmid on each reporter were arbitrarily set to 100 (fold activation was 72 and 74 for hb-CAT and kni-CAT , respectively). (C and D) CAT assay activities (in logarithmic scale) for wild-type Bcd and Bcd(92-489) on hb-CAT (C) and kni-CAT (D) reporters at different concentrations. The activities of wild-type Bcd at 1 μg of transfected effector DNA on each reporters were set to 100. (E) Representative Western blot results detecting Bcd proteins in transfected cells. For the experiments shown in this figure, the amounts of the transfected effector plasmids were 0.01, 0.03, 0.1, 0.3, and 1 μg. Wt, wild type.
    Figure Legend Snippet: The amino-terminal domain of Bcd plays different roles on hb and kni enhancer elements. (A) Schematic diagrams of the 250-bp hb enhancer element and the 64-bp kni enhancer element. The Bcd binding sites are represented by arrows. (B) CAT assay results of S2 cells transfected with hb-CAT and kni-CAT reporter plasmids (1 μg) and increasing amounts of an effector plasmid expressing Bcd. Activities obtained with 1 μg of transfected effector plasmid on each reporter were arbitrarily set to 100 (fold activation was 72 and 74 for hb-CAT and kni-CAT , respectively). (C and D) CAT assay activities (in logarithmic scale) for wild-type Bcd and Bcd(92-489) on hb-CAT (C) and kni-CAT (D) reporters at different concentrations. The activities of wild-type Bcd at 1 μg of transfected effector DNA on each reporters were set to 100. (E) Representative Western blot results detecting Bcd proteins in transfected cells. For the experiments shown in this figure, the amounts of the transfected effector plasmids were 0.01, 0.03, 0.1, 0.3, and 1 μg. Wt, wild type.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Expressing, Activation Assay, Western Blot

    The self-inhibitory function of Bcd is differentially implemented on hb and kni enhancers. (A) CAT assay results from S2 cells transfected with 1 μg of hb-CAT or kni-CAT reporter plasmid and 1 μg of effector plasmid expressing wild-type Bcd (lane 1), Bcd(92-489) (lane 2), Bcd(A52-56) (lane 3), or Bcd(A57-61) (lane 4). The activities of wild-type Bcd on each reporter were set to 100. The data for the hb-CAT ). Wt, wild type.
    Figure Legend Snippet: The self-inhibitory function of Bcd is differentially implemented on hb and kni enhancers. (A) CAT assay results from S2 cells transfected with 1 μg of hb-CAT or kni-CAT reporter plasmid and 1 μg of effector plasmid expressing wild-type Bcd (lane 1), Bcd(92-489) (lane 2), Bcd(A52-56) (lane 3), or Bcd(A57-61) (lane 4). The activities of wild-type Bcd on each reporter were set to 100. The data for the hb-CAT ). Wt, wild type.

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    6) Product Images from "Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K"

    Article Title: Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04090-2

    PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C
    Figure Legend Snippet: PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C

    Techniques Used: Activity Assay, Transfection, Construct, SDS Page, Mutagenesis, Clone Assay, Sequencing, Autoradiography, In Vitro, Kinase Assay, Staining, Western Blot, Immunostaining

    7) Product Images from "Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy"

    Article Title: Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy

    Journal: RNA

    doi: 10.1261/rna.057208.116

    snRNP-specific protein knockdown in S2 cells. ( A ) qRT-PCR of untreated cells versus those treated with dsRNA for  Smn ,  Prp6 , or  Prp8  mRNAs. ( B ) Western blot of dUTPase levels in RNAi-treated cells. Anti-SMN and anti-α-tubulin verify SMN knockdown
    Figure Legend Snippet: snRNP-specific protein knockdown in S2 cells. ( A ) qRT-PCR of untreated cells versus those treated with dsRNA for Smn , Prp6 , or Prp8 mRNAs. ( B ) Western blot of dUTPase levels in RNAi-treated cells. Anti-SMN and anti-α-tubulin verify SMN knockdown

    Techniques Used: Quantitative RT-PCR, Western Blot

    8) Product Images from "B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster"

    Article Title: B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.11.3674-3684.2002

    Apoptosis induced by loss of B56-regulated PP2A is accompanied by activation of DEVD-specific caspases and inhibited by RNAi of caspases Dredd, Dronc, and Drice. (A) Caspase activation following RNAi of B56-regulated PP2A. DEVD-specific caspase activity in the cell extracts was assayed 4 days after RNAi of the indicated genes. OD 405 , optical density at 405 nm. (B) Caspases Dredd, Dronc, and Drice are required for the cell death that follows RNAi of B56-targeted PP2A. Viable S2 cells were counted 4 days following RNAi of the indicated genes. (C to F) RNAi of dB56-1 and -2 (C) induces apoptotic morphology in S2 cells 4 days after application of the dsRNA, an effect blocked (D) by addition of dsRNA-targeting drice . Similar apoptotic changes were seen with RNAi of dIAP - 1 (E). Control cell morphology is shown in panel F.
    Figure Legend Snippet: Apoptosis induced by loss of B56-regulated PP2A is accompanied by activation of DEVD-specific caspases and inhibited by RNAi of caspases Dredd, Dronc, and Drice. (A) Caspase activation following RNAi of B56-regulated PP2A. DEVD-specific caspase activity in the cell extracts was assayed 4 days after RNAi of the indicated genes. OD 405 , optical density at 405 nm. (B) Caspases Dredd, Dronc, and Drice are required for the cell death that follows RNAi of B56-targeted PP2A. Viable S2 cells were counted 4 days following RNAi of the indicated genes. (C to F) RNAi of dB56-1 and -2 (C) induces apoptotic morphology in S2 cells 4 days after application of the dsRNA, an effect blocked (D) by addition of dsRNA-targeting drice . Similar apoptotic changes were seen with RNAi of dIAP - 1 (E). Control cell morphology is shown in panel F.

    Techniques Used: Activation Assay, Activity Assay

    RNAi-mediated knockdown suggests that PP2A is an obligate heterotrimer in S2 cells. Fifteen micrograms of each indicated dsRNA was added to S2 cells in six-well dishes as described (Materials and Methods), and the cognate proteins were examined by immunoblot analysis 96 h later. Dbt, the Drosophila CKIɛ homologue, was examined both as a control for equal protein loading and as a test of the specificity of RNAi. The dsRNA added is indicated along the top. −, no dsRNA added. The star indicates a cross-reacting band detected by the antibody directed against PR55. (A) RNAi of dPP2A A down-regulates A, C, and B subunits. (B) RNAi of dPP2A C down-regulates A, C, and B subunits. (C) RNAi of the four known B subunits in Drosophila down-regulates A and C subunits. (D) RNAi of dPP2A C has no effect on the mRNA levels of A and B subunits. Total RNA was extracted from S2 cells 4 days following RNAi of the indicated genes, followed by RT-PCR (RT) for A, C, B56-1, and Dbt. PCR products were separated by electrophoresis on 1% agarose and examined by ethidium bromide staining.
    Figure Legend Snippet: RNAi-mediated knockdown suggests that PP2A is an obligate heterotrimer in S2 cells. Fifteen micrograms of each indicated dsRNA was added to S2 cells in six-well dishes as described (Materials and Methods), and the cognate proteins were examined by immunoblot analysis 96 h later. Dbt, the Drosophila CKIɛ homologue, was examined both as a control for equal protein loading and as a test of the specificity of RNAi. The dsRNA added is indicated along the top. −, no dsRNA added. The star indicates a cross-reacting band detected by the antibody directed against PR55. (A) RNAi of dPP2A A down-regulates A, C, and B subunits. (B) RNAi of dPP2A C down-regulates A, C, and B subunits. (C) RNAi of the four known B subunits in Drosophila down-regulates A and C subunits. (D) RNAi of dPP2A C has no effect on the mRNA levels of A and B subunits. Total RNA was extracted from S2 cells 4 days following RNAi of the indicated genes, followed by RT-PCR (RT) for A, C, B56-1, and Dbt. PCR products were separated by electrophoresis on 1% agarose and examined by ethidium bromide staining.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Electrophoresis, Staining

    PP2A and its B56 regulatory subunits are required for S2 cell multiplication. Viable S2 cells per well (determined by trypan blue exclusion) were counted at days 1 to 4 after the addition of the indicated dsRNAs. The B56 subunits appear to be redundant for cell multiplication.
    Figure Legend Snippet: PP2A and its B56 regulatory subunits are required for S2 cell multiplication. Viable S2 cells per well (determined by trypan blue exclusion) were counted at days 1 to 4 after the addition of the indicated dsRNAs. The B56 subunits appear to be redundant for cell multiplication.

    Techniques Used:

    Loss of B56:PP2A induces apoptotic morphology in S2 cells. S2 cells were fixed and stained with DAPI 4 days after treatment. Nuclear morphology was examined after DAPI staining, and cell morphology was examined by phase-contrast microscopy. Cells were either mock treated (A) or treated with dsRNA from dPR55 (B), 40 μM actinomycin D for only 15 h (C), dsRNA from both B56-1 and B56-2 (dB56-1+2) (D), dPP2A A subunit (E), or dPP2A C subunit (F). PP2A A, C, and combined B56 subunits produced morphological changes similar to those induced by actinomycin D. No morphological changes were seen following RNAi of individual B56 subunits or of PR55 or PR72 either alone or combined (data not shown).
    Figure Legend Snippet: Loss of B56:PP2A induces apoptotic morphology in S2 cells. S2 cells were fixed and stained with DAPI 4 days after treatment. Nuclear morphology was examined after DAPI staining, and cell morphology was examined by phase-contrast microscopy. Cells were either mock treated (A) or treated with dsRNA from dPR55 (B), 40 μM actinomycin D for only 15 h (C), dsRNA from both B56-1 and B56-2 (dB56-1+2) (D), dPP2A A subunit (E), or dPP2A C subunit (F). PP2A A, C, and combined B56 subunits produced morphological changes similar to those induced by actinomycin D. No morphological changes were seen following RNAi of individual B56 subunits or of PR55 or PR72 either alone or combined (data not shown).

    Techniques Used: Staining, Microscopy, Produced

    B56-targeted PP2A acts functionally upstream of Dark/dApaf1, Reaper, and Hid, and dp53. (A) Loss of dark abolishes B56:PP2A-regulated activation of DEVD-dependent caspases. Caspase activity was determined 4 days following RNAi of the indicated genes. (B) RNAi of dPP2A C induces morphological changes characteristic of apoptosis. S2 cells were examined by phase-contrast microscopy 4 days following RNAi of dPP2A C. (C) Addition of dsRNA for dark blocks the morphology changes. Similar results were seen with dB56 subunits (data not shown). (D) Partial knockdown of upstream activators of apoptosis by RNAi in S2 cells. dsRNA derived from reaper , hid , grim , and dp53 was added to S2 cells as described earlier, and mRNA levels were assessed by RT-PCR 4 days later. RT-PCR (RT) against dbt was performed on each sample as a control for RNA recovery. (E) RNAi of rpr , hid , or dp53 but not of grim partially suppresses B56:PP2A-regulated activation of DEVD-specific caspases. OD 405 , optical density at 405 nm.
    Figure Legend Snippet: B56-targeted PP2A acts functionally upstream of Dark/dApaf1, Reaper, and Hid, and dp53. (A) Loss of dark abolishes B56:PP2A-regulated activation of DEVD-dependent caspases. Caspase activity was determined 4 days following RNAi of the indicated genes. (B) RNAi of dPP2A C induces morphological changes characteristic of apoptosis. S2 cells were examined by phase-contrast microscopy 4 days following RNAi of dPP2A C. (C) Addition of dsRNA for dark blocks the morphology changes. Similar results were seen with dB56 subunits (data not shown). (D) Partial knockdown of upstream activators of apoptosis by RNAi in S2 cells. dsRNA derived from reaper , hid , grim , and dp53 was added to S2 cells as described earlier, and mRNA levels were assessed by RT-PCR 4 days later. RT-PCR (RT) against dbt was performed on each sample as a control for RNA recovery. (E) RNAi of rpr , hid , or dp53 but not of grim partially suppresses B56:PP2A-regulated activation of DEVD-specific caspases. OD 405 , optical density at 405 nm.

    Techniques Used: Activation Assay, Activity Assay, Microscopy, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    9) Product Images from "N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs"

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.167338

    Drosophila sog  has three conserved glycosylation sites.  (A) Alignment of Sog sequences from  Drosophila sp . shows that three putative glycosylation sites are conserved (Arg residue in pink in  D. melanogaster ). A Tld/Tlr cleavage site sequence close to N1 is shown in red. (B) Sog protein scheme with the location of the four conserved CR domains, Tld/Tlr cleavage sites (red arrows) and putative glycosylation sites (pink). TM, transmembrane domain. (C-E) Analysis of Sog constructs expressed in S2 cells. (C) Glycosylation mutants produced by site-directed mutagenesis and their effect on Sog migration in SDS-PAGE. N23 and N123 migrate faster than wild-type Sog. All mutants and wild-type Sog bear a C-terminal V5/His tag. Treatment with Tunicamycin decreases wild-type Sog Mw and confirms that Sog is glycosylated. (D) SDS-PAGE for Sog protein in cells (C) and extracellular medium (M) shows that Sog is secreted and that only the N2 mutation slightly decreases Sog secretion. (E) ELISA for S2 cell-secreted Sog confirms the analysis in D.
    Figure Legend Snippet: Drosophila sog has three conserved glycosylation sites. (A) Alignment of Sog sequences from Drosophila sp . shows that three putative glycosylation sites are conserved (Arg residue in pink in D. melanogaster ). A Tld/Tlr cleavage site sequence close to N1 is shown in red. (B) Sog protein scheme with the location of the four conserved CR domains, Tld/Tlr cleavage sites (red arrows) and putative glycosylation sites (pink). TM, transmembrane domain. (C-E) Analysis of Sog constructs expressed in S2 cells. (C) Glycosylation mutants produced by site-directed mutagenesis and their effect on Sog migration in SDS-PAGE. N23 and N123 migrate faster than wild-type Sog. All mutants and wild-type Sog bear a C-terminal V5/His tag. Treatment with Tunicamycin decreases wild-type Sog Mw and confirms that Sog is glycosylated. (D) SDS-PAGE for Sog protein in cells (C) and extracellular medium (M) shows that Sog is secreted and that only the N2 mutation slightly decreases Sog secretion. (E) ELISA for S2 cell-secreted Sog confirms the analysis in D.

    Techniques Used: Sequencing, Construct, Produced, Mutagenesis, Migration, SDS Page, Enzyme-linked Immunosorbent Assay

    Loss of Sog glycosylation modifies Dpp binding. (A) S2 cells transfected with wild-type Sog-myc and different combinations of Tsg and BMP ligands. Co-immunoprecipitation (Co-IP) with cell supernatants shows that Sog binds only BMP heterodimers in the absence of Tsg, but binds Dpp homodimers in the presence of Tsg. (B) S2 cells transfected with wild-type Sog-myc or the N-terminal Supersog fragment reveal that only Supersog binds Dpp alone using cell pellets (Pellet). That Supersog interacts with Dpp in the extracellular space is confirmed by using cell supernatants (Sup). The presence of Sax or Tkv in lane 3 does not change this pattern. (C) S2 cells transfected with wild-type sog- V5 or sogN123 -V5, and dpp- HA. Co-IP for V5 suggests that binding of the N123 mutant to Dpp is weakly enhanced compared with wild-type Sog. (D) S2 cells transfected with wild-type sog (S) or single glycosylation mutants (N1, N2, N3) plus dpp -HA, or plus dpp- HA and tld -HA. Co-IP for V5. SogN1 binds weakly to Dpp alone, whereas other mutants and wild-type Sog do not. Arrows in D point to the different Sog fragments produced by the Tld metalloprotease and show that the cleavage pattern is similar among all constructs. Asterisks indicate nonspecific bands.
    Figure Legend Snippet: Loss of Sog glycosylation modifies Dpp binding. (A) S2 cells transfected with wild-type Sog-myc and different combinations of Tsg and BMP ligands. Co-immunoprecipitation (Co-IP) with cell supernatants shows that Sog binds only BMP heterodimers in the absence of Tsg, but binds Dpp homodimers in the presence of Tsg. (B) S2 cells transfected with wild-type Sog-myc or the N-terminal Supersog fragment reveal that only Supersog binds Dpp alone using cell pellets (Pellet). That Supersog interacts with Dpp in the extracellular space is confirmed by using cell supernatants (Sup). The presence of Sax or Tkv in lane 3 does not change this pattern. (C) S2 cells transfected with wild-type sog- V5 or sogN123 -V5, and dpp- HA. Co-IP for V5 suggests that binding of the N123 mutant to Dpp is weakly enhanced compared with wild-type Sog. (D) S2 cells transfected with wild-type sog (S) or single glycosylation mutants (N1, N2, N3) plus dpp -HA, or plus dpp- HA and tld -HA. Co-IP for V5. SogN1 binds weakly to Dpp alone, whereas other mutants and wild-type Sog do not. Arrows in D point to the different Sog fragments produced by the Tld metalloprotease and show that the cleavage pattern is similar among all constructs. Asterisks indicate nonspecific bands.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Produced, Construct

    Sog levels are controlled by glycan and receptor-based retrieval from the extracellular space. (A) S2 cells transfected with wild-type sog- myc construct and different combinations of constructs producing Tsg and BMP ligands. Cellular levels of Sog are detected with anti-myc antiserum. Sog amounts increase in the presence of Dpp plus Tsg. These cellular levels correspond to Sog retrieved from the medium, as equivalent intracellular levels are observed when medium (S) from construct-expressing cells is added to cells that do not express sog- myc . (B) Intracellular Sog levels retrieved from Sog-myc+Dpp-HA+Tsg-His medium decrease by knocking down the Dpp receptor gene tkv or αPS1 ( mew ), αPS2 ( if ) or βPS ( mys ) integrin expression. (C) S2 cells transfected with wild-type sog-V5 or sogN1-V5 , sogN2-V5 , sogN3-V5 or triple sogN123-V5 mutant constructs in the presence of constructs producing Tsg and Dpp. Sog retrieval decreases by mutating the N1 and N2 sites. (D) Predicted conformation of Sog protein with regions depicted for binding to established extracellular partners. Red arrows indicate Tld/Tlr cleavage sites; pink represents glycosylated residues; gray shaded and open circles indicate BMPs. (E-J) Medium from S2 cells transfected with dpp -HA and tsg -His plus wild-type sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 was added to naive cells and immunolabeled with anti-V5 (red) and anti-Rab5 (green) (E-H′). (E′′) High magnification for wild-type Sog-V5 and Rab5 shows partial colocalization, indicative of Sog endocytosis. Arrows indicate V5 + Rab5 + punctae. Quantification of these cells (I) shows that loss of N1, N2 and N3 glycosylation sites decreases the number of cells with V5 + Rab5 + endocytic punctae. (J) In cells expressing dpp- HA , gbb- His and tsg- His plus wild-type (wt) sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 , the number of cells with V5 + Rab5 + punctae is equivalent. ** P ≤0.01 (Student's t -test). Scale bars: 20 μm in E′; 10 μm in E″.
    Figure Legend Snippet: Sog levels are controlled by glycan and receptor-based retrieval from the extracellular space. (A) S2 cells transfected with wild-type sog- myc construct and different combinations of constructs producing Tsg and BMP ligands. Cellular levels of Sog are detected with anti-myc antiserum. Sog amounts increase in the presence of Dpp plus Tsg. These cellular levels correspond to Sog retrieved from the medium, as equivalent intracellular levels are observed when medium (S) from construct-expressing cells is added to cells that do not express sog- myc . (B) Intracellular Sog levels retrieved from Sog-myc+Dpp-HA+Tsg-His medium decrease by knocking down the Dpp receptor gene tkv or αPS1 ( mew ), αPS2 ( if ) or βPS ( mys ) integrin expression. (C) S2 cells transfected with wild-type sog-V5 or sogN1-V5 , sogN2-V5 , sogN3-V5 or triple sogN123-V5 mutant constructs in the presence of constructs producing Tsg and Dpp. Sog retrieval decreases by mutating the N1 and N2 sites. (D) Predicted conformation of Sog protein with regions depicted for binding to established extracellular partners. Red arrows indicate Tld/Tlr cleavage sites; pink represents glycosylated residues; gray shaded and open circles indicate BMPs. (E-J) Medium from S2 cells transfected with dpp -HA and tsg -His plus wild-type sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 was added to naive cells and immunolabeled with anti-V5 (red) and anti-Rab5 (green) (E-H′). (E′′) High magnification for wild-type Sog-V5 and Rab5 shows partial colocalization, indicative of Sog endocytosis. Arrows indicate V5 + Rab5 + punctae. Quantification of these cells (I) shows that loss of N1, N2 and N3 glycosylation sites decreases the number of cells with V5 + Rab5 + endocytic punctae. (J) In cells expressing dpp- HA , gbb- His and tsg- His plus wild-type (wt) sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 , the number of cells with V5 + Rab5 + punctae is equivalent. ** P ≤0.01 (Student's t -test). Scale bars: 20 μm in E′; 10 μm in E″.

    Techniques Used: Transfection, Construct, Expressing, Mutagenesis, Binding Assay, Immunolabeling

    10) Product Images from "The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth"

    Article Title: The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

    Journal: bioRxiv

    doi: 10.1101/2021.09.06.459106

    Nord binds to Dpp and attenuates BMP signaling in vitro (A) Co-immunoprecipitation of Nord with the BMP ligands Dpp and Gbb. Medium from S2 cells transfected for expression of GFP-tagged Nord were mixed with medium from cells expressing Flag-tagged Dpp and HA-tagged Gbb alone or in combination, followed by incubation with anti-FLAG or anti-HA antibody coupled beads overnight at 4°C. Precipitated proteins were analyzed by Western blotting with indicated antibodies. Nord was immunoprecipitated with Dpp and, to a lesser extent, with Gbb. The amount of immunoprecipitated Nord was increased when Dpp and Gbb were co-transfected. (B) S2 cells were transfected for expression of Flag-tagged Dpp or Gbb with or without GFP-tagged Nord (source cell). Both cell lysate and conditioned medium from the source cells were collected and followed by Western blot analysis. Loading was controlled by probing the blot for tubulin. The amount of Dpp or Dpp-Gbb ligands released into the medium was reduced when Nord was co-expressed in the source cells. (C) Comparison of BMP signaling activities of conditioned media in a cell-based signaling assay. After incubating the conditioned media collected in (B) with S2 cells stably expressing the FLAG-Mad transgene (Mad-S2, responding cell) for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (D) Quantification of the Western blot data in panel C: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (E) Quantification of relative ligand activity [(pMad/Mad)/BMPs] by normalizing the medium signaling activity pMad/Mad in panel C to the ligand amount in panel B. (F) Mad-S2 cells were treated with a recombinant Dpp peptide (rDpp) in the absence or presence of conditioned medium containing raising levels of Nord for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (G) Quantification of the Western blot data in panel F: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (H) Mad-S2 cells were transiently transfected for expression of GFP or Nord-GFP. 48 hours after transfection, the cells were treated with recombinant Dpp peptides for 1 hour. Upon treatment, the cells were washed, fixed, and stained by anti-FLAG to detect total Mad and anti-pMad to detect phosphorylated Mad. The average pMad levels were measured and normalized to the total Mad levels, and then plotted against different Dpp concentrations (0 to 5 nM). Each point shows the mean ± SD, n > 10. au: arbitrary units. The unpaired two-tailed t-test was used for statistical analysis. ***P
    Figure Legend Snippet: Nord binds to Dpp and attenuates BMP signaling in vitro (A) Co-immunoprecipitation of Nord with the BMP ligands Dpp and Gbb. Medium from S2 cells transfected for expression of GFP-tagged Nord were mixed with medium from cells expressing Flag-tagged Dpp and HA-tagged Gbb alone or in combination, followed by incubation with anti-FLAG or anti-HA antibody coupled beads overnight at 4°C. Precipitated proteins were analyzed by Western blotting with indicated antibodies. Nord was immunoprecipitated with Dpp and, to a lesser extent, with Gbb. The amount of immunoprecipitated Nord was increased when Dpp and Gbb were co-transfected. (B) S2 cells were transfected for expression of Flag-tagged Dpp or Gbb with or without GFP-tagged Nord (source cell). Both cell lysate and conditioned medium from the source cells were collected and followed by Western blot analysis. Loading was controlled by probing the blot for tubulin. The amount of Dpp or Dpp-Gbb ligands released into the medium was reduced when Nord was co-expressed in the source cells. (C) Comparison of BMP signaling activities of conditioned media in a cell-based signaling assay. After incubating the conditioned media collected in (B) with S2 cells stably expressing the FLAG-Mad transgene (Mad-S2, responding cell) for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (D) Quantification of the Western blot data in panel C: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (E) Quantification of relative ligand activity [(pMad/Mad)/BMPs] by normalizing the medium signaling activity pMad/Mad in panel C to the ligand amount in panel B. (F) Mad-S2 cells were treated with a recombinant Dpp peptide (rDpp) in the absence or presence of conditioned medium containing raising levels of Nord for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (G) Quantification of the Western blot data in panel F: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (H) Mad-S2 cells were transiently transfected for expression of GFP or Nord-GFP. 48 hours after transfection, the cells were treated with recombinant Dpp peptides for 1 hour. Upon treatment, the cells were washed, fixed, and stained by anti-FLAG to detect total Mad and anti-pMad to detect phosphorylated Mad. The average pMad levels were measured and normalized to the total Mad levels, and then plotted against different Dpp concentrations (0 to 5 nM). Each point shows the mean ± SD, n > 10. au: arbitrary units. The unpaired two-tailed t-test was used for statistical analysis. ***P

    Techniques Used: In Vitro, Immunoprecipitation, Transfection, Expressing, Incubation, Western Blot, Stable Transfection, Activity Assay, Recombinant, Staining, Two Tailed Test

    11) Product Images from "Enhanced antimicrobial peptide-induced activity in the mollusc Toll-2 family through evolution via tandem Toll/interleukin-1 receptor"

    Article Title: Enhanced antimicrobial peptide-induced activity in the mollusc Toll-2 family through evolution via tandem Toll/interleukin-1 receptor

    Journal: Royal Society Open Science

    doi: 10.1098/rsos.160123

    Activation of shrimp  Pen4  antimicrobial peptide gene by over expression of the full length of  Hcu_Toll-I  and  Hcu_Toll-2-2  ( a ),  Hcu_Toll-2-2 -TIR-1,  Hcu_Toll-2-2 -TIR-2 and  Hcu_Toll-2-2 -TIR-1 + TIR-2 ( b ) in S2 cells. The detailed methods could be seen in Material and methods section. All data are representative of three independent experiments. The bars indicate the s.d. of the luciferase activity ( n  = 3).
    Figure Legend Snippet: Activation of shrimp Pen4 antimicrobial peptide gene by over expression of the full length of Hcu_Toll-I and Hcu_Toll-2-2 ( a ), Hcu_Toll-2-2 -TIR-1, Hcu_Toll-2-2 -TIR-2 and Hcu_Toll-2-2 -TIR-1 + TIR-2 ( b ) in S2 cells. The detailed methods could be seen in Material and methods section. All data are representative of three independent experiments. The bars indicate the s.d. of the luciferase activity ( n  = 3).

    Techniques Used: Activation Assay, Over Expression, Luciferase, Activity Assay

    12) Product Images from "Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit"

    Article Title: Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002620

    D. melanogaster RpS9 is autoregulated by alternative splicing coupled to NMD. A) Illustration of RpS9 mRNA isoforms assessed by PCR (the PTC+ isoform is indicated by a red octagon). Primers sets (arrows) were designed to amplify multiple or specific RpS9 mRNA isoforms (RT-PCR and qPCR primers, respectively). B) RT-PCR validation of the RpS9 PTC+ mRNA isoform degraded by NMD. C) Experimental design used to assess the affect of UPF1 knock-down on the abundance of RpS9 mRNA isoforms. D) RT-qPCR determination of RpS9 PTC+ mRNA isoform abundance (top panels) and total endogenous RpS9 mRNA abundance (bottom panels) in S2 cells transfected with a plasmid constitutively expressing an RpS9 cDNA (red circles) or an empty vector control (blue circles). The affect of UPF1 knock-down (via incubation with dsRNA) on each RpS9 mRNA isoform (right panels) is compared to a non-specific dsRNA control (left panels). RpS9 mRNA isoform abundance values were divided by GAPDH1 mRNA abundance values to obtain ratios internally controlled for variations in cDNA quantity. Log 2 transformed ratios for each of three biological replicates is shown as a point and the mean as a dash.
    Figure Legend Snippet: D. melanogaster RpS9 is autoregulated by alternative splicing coupled to NMD. A) Illustration of RpS9 mRNA isoforms assessed by PCR (the PTC+ isoform is indicated by a red octagon). Primers sets (arrows) were designed to amplify multiple or specific RpS9 mRNA isoforms (RT-PCR and qPCR primers, respectively). B) RT-PCR validation of the RpS9 PTC+ mRNA isoform degraded by NMD. C) Experimental design used to assess the affect of UPF1 knock-down on the abundance of RpS9 mRNA isoforms. D) RT-qPCR determination of RpS9 PTC+ mRNA isoform abundance (top panels) and total endogenous RpS9 mRNA abundance (bottom panels) in S2 cells transfected with a plasmid constitutively expressing an RpS9 cDNA (red circles) or an empty vector control (blue circles). The affect of UPF1 knock-down (via incubation with dsRNA) on each RpS9 mRNA isoform (right panels) is compared to a non-specific dsRNA control (left panels). RpS9 mRNA isoform abundance values were divided by GAPDH1 mRNA abundance values to obtain ratios internally controlled for variations in cDNA quantity. Log 2 transformed ratios for each of three biological replicates is shown as a point and the mean as a dash.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Incubation, Transformation Assay

    13) Product Images from "Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K"

    Article Title: Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04090-2

    PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C
    Figure Legend Snippet: PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C

    Techniques Used: Activity Assay, Transfection, Construct, SDS Page, Mutagenesis, Clone Assay, Sequencing, Autoradiography, In Vitro, Kinase Assay, Staining, Western Blot, Immunostaining

    14) Product Images from "Post-mitotic myotubes repurpose the cytokinesis machinery to effect cellular guidance"

    Article Title: Post-mitotic myotubes repurpose the cytokinesis machinery to effect cellular guidance

    Journal: bioRxiv

    doi: 10.1101/2020.06.16.155333

    related to Figure 4. (A) Coomassie stained control gel for GST immunoprecipitation experiment described in Figure 3 . Bsd-bound GST beads (lane 5) recovered a large number of unique bands compared to control GST beads (lane 4). (B) Co-immunoprecipitation screen. S2 cells were transfected with Bsd.Myc and either a control plasmid (lane 2) or a plasmid expressing a Flag-tagged ‘prey’ protein (lanes 3-8) identified by AP-MS. Polo showed the strongest interaction with Bsd among the proteins tested. Lane 1 is a positive control. (C) Muscle morphogenesis phenotypes in polo mutants. Stage 16 embryos labeled with Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. The polo alleles showed stronger phenotypes showed stronger phenotypes in trans to Df(3L)BSC447 that uncovers polo , arguing the alleles are hypomorphic. Quantification of muscle phenotypes is as described in Fig. 1 .
    Figure Legend Snippet: related to Figure 4. (A) Coomassie stained control gel for GST immunoprecipitation experiment described in Figure 3 . Bsd-bound GST beads (lane 5) recovered a large number of unique bands compared to control GST beads (lane 4). (B) Co-immunoprecipitation screen. S2 cells were transfected with Bsd.Myc and either a control plasmid (lane 2) or a plasmid expressing a Flag-tagged ‘prey’ protein (lanes 3-8) identified by AP-MS. Polo showed the strongest interaction with Bsd among the proteins tested. Lane 1 is a positive control. (C) Muscle morphogenesis phenotypes in polo mutants. Stage 16 embryos labeled with Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. The polo alleles showed stronger phenotypes showed stronger phenotypes in trans to Df(3L)BSC447 that uncovers polo , arguing the alleles are hypomorphic. Quantification of muscle phenotypes is as described in Fig. 1 .

    Techniques Used: Staining, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Positive Control, Labeling

    related to Figure 5. (A) Control phosphorylation assay. Lysates from S2 cells transfected with a control plasmid or a plasmid expressing Bsd.Myc were immunoblotted with anti-phospho-threonine (pThr). Whole lystates showed equivalent pThr levels. (B) in vitro phosphorylation assay. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd.I129A showed equivalent phosphorylated Polo as controls. (C) Stage 12 embryos with an endogenous GFP-tagged Polo ( Polo GFP ) that expressed rp298 > nLacZ were immunolabeled for GFP (green) and lacZ (red). Localization of total Polo protein was comparable between control and bsd 1 embryos. Graph shows Polo and nlacZ fluorescent intensity across myoblasts. (D) Early and late Stage 12 embryos immunolabeled for phospo-Polo (pPolo, green) and Mef2 (violet). pPolo localization in myonuclei increased during Stage 12 in control ( bsd 1 heterozygous) but not bsd 1 embryos. Control and bsd 1 embryos were labeled in the same preparation. (E) Immunoprecipitation of S2 cell lysates transfected with Bsd and Pav. Bsd did not physically interact with Pav.
    Figure Legend Snippet: related to Figure 5. (A) Control phosphorylation assay. Lysates from S2 cells transfected with a control plasmid or a plasmid expressing Bsd.Myc were immunoblotted with anti-phospho-threonine (pThr). Whole lystates showed equivalent pThr levels. (B) in vitro phosphorylation assay. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd.I129A showed equivalent phosphorylated Polo as controls. (C) Stage 12 embryos with an endogenous GFP-tagged Polo ( Polo GFP ) that expressed rp298 > nLacZ were immunolabeled for GFP (green) and lacZ (red). Localization of total Polo protein was comparable between control and bsd 1 embryos. Graph shows Polo and nlacZ fluorescent intensity across myoblasts. (D) Early and late Stage 12 embryos immunolabeled for phospo-Polo (pPolo, green) and Mef2 (violet). pPolo localization in myonuclei increased during Stage 12 in control ( bsd 1 heterozygous) but not bsd 1 embryos. Control and bsd 1 embryos were labeled in the same preparation. (E) Immunoprecipitation of S2 cell lysates transfected with Bsd and Pav. Bsd did not physically interact with Pav.

    Techniques Used: Phosphorylation Assay, Transfection, Plasmid Preparation, Expressing, In Vitro, Immunoprecipitation, Immunolabeling, Labeling

    Bsd activates Polo to regulate myotube guidance. (A)  in vitro  phosphorylation assays. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd showed more phosphorylated Polo than controls. (B) Polo localization in S2 cells. Bsd.Myc, Polo.Flag, and Polo.T182A.Flag were transfected into S2 cells; transgenic proteins were detected with anti-Myc (red, Bsd) and anti-Flag (green, Polo). Polo localized to discrete nuclear foci in a subset of control cells (middle column). The frequency of cells showing nuclear Polo foci was increased when Bsd was co-transfected with Polo (left column and graph). Cells transfected with Polo that lacked a phosphorylated threonine (T182A) showed no nuclear Polo foci (right column and graph). (C) Polo acts downstream of Bsd during myogenesis.  bsd 1  UAS.Polo.T182D  and  bsd 1  Mef2.Gal4 > Polo.T182D  embryos labeled for Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. Expressing active (phosphomimetic) Polo T182D  in the mesoderm of  bsd 1  embryos suppressed the  bsd 1  myogenic phenotype. Quantification of myogenic phenotypes is as described in   Fig. 1 . (D)  in vivo  phosphorylation assays. Endogenous GFP-tagged Polo was immunoprecipitated from embryo lysates and blotted with pThr.  bsd 1  embryo lysates showed significantly less phosphorylated Polo than controls. (E) Stage 12 embryos immunolabeled for phosphorylated Polo (Polo pT182 , green) and Mef2 (violet). Polo pT182  localized to myonuclei in control embryos. (F) Polo pT182  levels in myonuclei were significantly reduced in  bsd 1  embryos. (G)  tum DH15  VL1, DT1, LO1, and VT1 muscle phenotypes. Stage 16 embryos labeled for  5053 > GFP  (green) and Tropomyosin (violet) or  slou > GFP  (green) and Tropomyosin (violet).  tum DH15  muscles made incorrect tendon attachments similar to  bsd 1  muscles (white arrowheads). (H) Histogram of VL1, DT1, LO1, and VT1 phenotypes (n≥54 per muscle). (ns) not significant, **(p
    Figure Legend Snippet: Bsd activates Polo to regulate myotube guidance. (A) in vitro phosphorylation assays. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd showed more phosphorylated Polo than controls. (B) Polo localization in S2 cells. Bsd.Myc, Polo.Flag, and Polo.T182A.Flag were transfected into S2 cells; transgenic proteins were detected with anti-Myc (red, Bsd) and anti-Flag (green, Polo). Polo localized to discrete nuclear foci in a subset of control cells (middle column). The frequency of cells showing nuclear Polo foci was increased when Bsd was co-transfected with Polo (left column and graph). Cells transfected with Polo that lacked a phosphorylated threonine (T182A) showed no nuclear Polo foci (right column and graph). (C) Polo acts downstream of Bsd during myogenesis. bsd 1 UAS.Polo.T182D and bsd 1 Mef2.Gal4 > Polo.T182D embryos labeled for Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. Expressing active (phosphomimetic) Polo T182D in the mesoderm of bsd 1 embryos suppressed the bsd 1 myogenic phenotype. Quantification of myogenic phenotypes is as described in Fig. 1 . (D) in vivo phosphorylation assays. Endogenous GFP-tagged Polo was immunoprecipitated from embryo lysates and blotted with pThr. bsd 1 embryo lysates showed significantly less phosphorylated Polo than controls. (E) Stage 12 embryos immunolabeled for phosphorylated Polo (Polo pT182 , green) and Mef2 (violet). Polo pT182 localized to myonuclei in control embryos. (F) Polo pT182 levels in myonuclei were significantly reduced in bsd 1 embryos. (G) tum DH15 VL1, DT1, LO1, and VT1 muscle phenotypes. Stage 16 embryos labeled for 5053 > GFP (green) and Tropomyosin (violet) or slou > GFP (green) and Tropomyosin (violet). tum DH15 muscles made incorrect tendon attachments similar to bsd 1 muscles (white arrowheads). (H) Histogram of VL1, DT1, LO1, and VT1 phenotypes (n≥54 per muscle). (ns) not significant, **(p

    Techniques Used: In Vitro, Immunoprecipitation, Transfection, Transgenic Assay, Labeling, Expressing, In Vivo, Immunolabeling

    15) Product Images from "The Shrimp NF-?B Pathway Is Activated by White Spot Syndrome Virus (WSSV) 449 to Facilitate the Expression of WSSV069 (ie1), WSSV303 and WSSV371"

    Article Title: The Shrimp NF-?B Pathway Is Activated by White Spot Syndrome Virus (WSSV) 449 to Facilitate the Expression of WSSV069 (ie1), WSSV303 and WSSV371

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024773

    A promoter screen to identify viral genes induced by NF-κB activation. (A) The determination of the promoter activities of 40 WSSV genes when the shrimp NF-κB family protein LvDorsal is overexpressed in S2 cells. All of the 40 WSSV genes possess NF-κB binding sites in their promoter regions. The promoter regions were inserted into pGL3-Basic to construct luciferase reporters. When transfected into S2 cells, the promoters of WSSV069 ( ie1 ), WSSV303 and WSSV371 are activated by LvDorsal. (B) Stimulated by WSSV, LvDorsal translocated to the nucleus. PBS treated cells were used as a control. (C) The promoter regions containing NF-κB binding sites of WSSV069 ( ie1 ), WSSV303 and WSSV371 . The NF-κB binding sites were in bold and underlined.
    Figure Legend Snippet: A promoter screen to identify viral genes induced by NF-κB activation. (A) The determination of the promoter activities of 40 WSSV genes when the shrimp NF-κB family protein LvDorsal is overexpressed in S2 cells. All of the 40 WSSV genes possess NF-κB binding sites in their promoter regions. The promoter regions were inserted into pGL3-Basic to construct luciferase reporters. When transfected into S2 cells, the promoters of WSSV069 ( ie1 ), WSSV303 and WSSV371 are activated by LvDorsal. (B) Stimulated by WSSV, LvDorsal translocated to the nucleus. PBS treated cells were used as a control. (C) The promoter regions containing NF-κB binding sites of WSSV069 ( ie1 ), WSSV303 and WSSV371 . The NF-κB binding sites were in bold and underlined.

    Techniques Used: Activation Assay, Binding Assay, Construct, Luciferase, Transfection

    The functional study of LvPelle in the Toll pathway. (A) The intracellular localization of LvPelle and its truncated mutants fused with GFP in S2 cells. P1 represented amino acids 1–69 of LvPelle, P2 represented amino acids 1–262 of LvPelle, P3 represented amino acids 1–536 of LvPelle (the full-length protein of LvPelle) and P4 represented amino acids 129–536 of LvPelle as indicated in   Fig. 2 . (B) Overexpression of LvPelle activates  Drosophila  and shrimp AMP promoters. Luciferase reporter genes including pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs and pGL3-AttA were constructed successfully and demonstrated to be predominantly regulated through NF-κB activation   [34] ,   [35] ,   [37] ,   [38] ,   [39] ,   [40] . In this study, we use these luciferase reporter genes to investigate the activation of Toll-mediated NF-κB pathway. The data are representative of three independent experiments. **p
    Figure Legend Snippet: The functional study of LvPelle in the Toll pathway. (A) The intracellular localization of LvPelle and its truncated mutants fused with GFP in S2 cells. P1 represented amino acids 1–69 of LvPelle, P2 represented amino acids 1–262 of LvPelle, P3 represented amino acids 1–536 of LvPelle (the full-length protein of LvPelle) and P4 represented amino acids 129–536 of LvPelle as indicated in Fig. 2 . (B) Overexpression of LvPelle activates Drosophila and shrimp AMP promoters. Luciferase reporter genes including pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs and pGL3-AttA were constructed successfully and demonstrated to be predominantly regulated through NF-κB activation [34] , [35] , [37] , [38] , [39] , [40] . In this study, we use these luciferase reporter genes to investigate the activation of Toll-mediated NF-κB pathway. The data are representative of three independent experiments. **p

    Techniques Used: Functional Assay, Over Expression, Luciferase, Construct, Activation Assay

    WSSV449 and LvPelle activate the promoters of WSSV069 ( ie1 ), WSSV303 and WSSV371 . The S2 cells were transfected with 0.05 µg of protein expression vector (pAC5.1-LvPelle or pAC5.1-WSSV449), with 0.05 µg reporter gene plasmid (pGL3-Bsic, pGL3-WSSV069, pGL3-WSSV303 or pGL3-WSSV371) and with 0.005 µg pRL-TK Renilla luciferase plasmid (as an internal control, Promega, USA). Thirty-six hours after transfection, the cells were harvested and analyzed using the Dual Luciferase kit. The data are representative of three independent experiments. **p
    Figure Legend Snippet: WSSV449 and LvPelle activate the promoters of WSSV069 ( ie1 ), WSSV303 and WSSV371 . The S2 cells were transfected with 0.05 µg of protein expression vector (pAC5.1-LvPelle or pAC5.1-WSSV449), with 0.05 µg reporter gene plasmid (pGL3-Bsic, pGL3-WSSV069, pGL3-WSSV303 or pGL3-WSSV371) and with 0.005 µg pRL-TK Renilla luciferase plasmid (as an internal control, Promega, USA). Thirty-six hours after transfection, the cells were harvested and analyzed using the Dual Luciferase kit. The data are representative of three independent experiments. **p

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase

    16) Product Images from "Requirement for a Nuclear Function of ?-Catenin in Wnt Signaling"

    Article Title: Requirement for a Nuclear Function of ?-Catenin in Wnt Signaling

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.23.8462-8470.2003

    Requirement of endogenous β-catenin for the stimulatory effect of membrane-tethered β-catenin/plakoglobin on luciferase reporters. (A) Effect of β-catenin-specific siRNA on the expression of endogenous β-catenin. 293 cells or NIH 3T3 cells were treated with control (c) and human or mouse-specific β-catenin (hβ or mβ) siRNA, and the expression of endogenous β-catenin was determined by immunoblotting with anti-β-catenin antibodies. Equal loading was confirmed by Western blotting with anti-α-tubulin antibodies. (B) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered plakoglobin (CnxPkg) in 293 cells. 293 cells were transfected with control (▪) or human β-catenin-specific ( ) siRNA, together with TOP-FLASH and the indicated expression constructs, and the luciferase activities were determined 48 h after transfection. (C) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered β-catenin in NIH 3T3 cells. To achieve high sensitivity in NIH 3T3 cells, β-catenin was cotransfected with a mouse LEF-1 expression plasmid and a LEF-1 luciferase reporter that contains multiple LEF-1 binding sites. NIH 3T3 cells were transfected with control (▪) or mouse β-catenin-specific ( ) siRNA, together with the indicated expression constructs, and assayed for luciferase activity. Amino acid residues 675 to 781 of β-catenin were deleted from Cnxβ to form CnxβΔC. (D) Requirement of endogenous armadillo for the signaling activity of membrane-tethered β-catenin in Drosophila S2 cells. S2 cells were grown in six-well plates and treated with control (▪) or armadillo-specific ( ) dsRNA at a concentration of 15 μg/well. Cells were then transfected with LEF-luciferase and LEF-1 expression constructs with the indicated plasmids that encode various forms of plakoglobin and β-catenin. Amino acid residues 677 to 781 and 583 to 781 of β-catenin were removed from Cnxβ to form CnxβΔC1 and CnxβΔC2. (E) Requirement of endogenous β-catenin for membrane-tethered plakoglobin-induced DKK1 expression. 293 cells were transfected with control siRNA (▪) or human β-catenin-specific siRNA ( ) with the indicated expression plasmids. The expression of DKK1 gene was determined by quantitative real-time PCR after reverse transcription. vec, Vector.
    Figure Legend Snippet: Requirement of endogenous β-catenin for the stimulatory effect of membrane-tethered β-catenin/plakoglobin on luciferase reporters. (A) Effect of β-catenin-specific siRNA on the expression of endogenous β-catenin. 293 cells or NIH 3T3 cells were treated with control (c) and human or mouse-specific β-catenin (hβ or mβ) siRNA, and the expression of endogenous β-catenin was determined by immunoblotting with anti-β-catenin antibodies. Equal loading was confirmed by Western blotting with anti-α-tubulin antibodies. (B) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered plakoglobin (CnxPkg) in 293 cells. 293 cells were transfected with control (▪) or human β-catenin-specific ( ) siRNA, together with TOP-FLASH and the indicated expression constructs, and the luciferase activities were determined 48 h after transfection. (C) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered β-catenin in NIH 3T3 cells. To achieve high sensitivity in NIH 3T3 cells, β-catenin was cotransfected with a mouse LEF-1 expression plasmid and a LEF-1 luciferase reporter that contains multiple LEF-1 binding sites. NIH 3T3 cells were transfected with control (▪) or mouse β-catenin-specific ( ) siRNA, together with the indicated expression constructs, and assayed for luciferase activity. Amino acid residues 675 to 781 of β-catenin were deleted from Cnxβ to form CnxβΔC. (D) Requirement of endogenous armadillo for the signaling activity of membrane-tethered β-catenin in Drosophila S2 cells. S2 cells were grown in six-well plates and treated with control (▪) or armadillo-specific ( ) dsRNA at a concentration of 15 μg/well. Cells were then transfected with LEF-luciferase and LEF-1 expression constructs with the indicated plasmids that encode various forms of plakoglobin and β-catenin. Amino acid residues 677 to 781 and 583 to 781 of β-catenin were removed from Cnxβ to form CnxβΔC1 and CnxβΔC2. (E) Requirement of endogenous β-catenin for membrane-tethered plakoglobin-induced DKK1 expression. 293 cells were transfected with control siRNA (▪) or human β-catenin-specific siRNA ( ) with the indicated expression plasmids. The expression of DKK1 gene was determined by quantitative real-time PCR after reverse transcription. vec, Vector.

    Techniques Used: Luciferase, Expressing, Western Blot, Activity Assay, Transfection, Construct, Plasmid Preparation, Binding Assay, Concentration Assay, Real-time Polymerase Chain Reaction

    17) Product Images from "Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila"

    Article Title: Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000670

    The genes corresponding to transcript targets of HP1a in S2 cells show a good overlap with HP1a binding sites on polytene chromosomes and appear down-regulated in both S2 cells lacking HP1a and HP1a mutant larvae. (A) Localization of HP1a binding sites and the genes corresponding to the HP1a target transcripts along polytene chromosomes of Drosophila wild type larvae. Blue bars represent sites where the HP1a target genes overlap with HP1a immunosignals; orange bars indicate the localization of HP1a target genes that do not overlap with HP1a immunosignals. (B) Quantitative RT–PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of target genes that overlap with HP1a on polytene chromosomes whose position is indicated in (A) by blue bars marked with asterisks and (C) a sub-set of HP1a target genes that do not overlap with HP1a on polytene chromosomes whose position is indicated in (A) by orange bars marked with asterisks. (D) Quantitative RT-PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of genes that were not found among the HP1a target genes in S2 cells and do not co-map with any of the HP1a immunosignals along the polytene chromosomes. (E) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2B . (F) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2C . (G) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of non target genes reported in Figure 2D .
    Figure Legend Snippet: The genes corresponding to transcript targets of HP1a in S2 cells show a good overlap with HP1a binding sites on polytene chromosomes and appear down-regulated in both S2 cells lacking HP1a and HP1a mutant larvae. (A) Localization of HP1a binding sites and the genes corresponding to the HP1a target transcripts along polytene chromosomes of Drosophila wild type larvae. Blue bars represent sites where the HP1a target genes overlap with HP1a immunosignals; orange bars indicate the localization of HP1a target genes that do not overlap with HP1a immunosignals. (B) Quantitative RT–PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of target genes that overlap with HP1a on polytene chromosomes whose position is indicated in (A) by blue bars marked with asterisks and (C) a sub-set of HP1a target genes that do not overlap with HP1a on polytene chromosomes whose position is indicated in (A) by orange bars marked with asterisks. (D) Quantitative RT-PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of genes that were not found among the HP1a target genes in S2 cells and do not co-map with any of the HP1a immunosignals along the polytene chromosomes. (E) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2B . (F) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2C . (G) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of non target genes reported in Figure 2D .

    Techniques Used: Binding Assay, Mutagenesis, Quantitative RT-PCR, Expressing

    Genes corresponding to transcript targets of HP1a in S2 cells appear down-regulated also in DDP1 and PEP mutants larvae. (A) Quantitative RT–PCR analysis of the expression in wild type Ore-R and DDP1 mutant larvae showing a significant decrease in the amount of the transcripts corresponding to all genes. (B) Quantitative RT–PCR analysis in wild type Ore-R and PEP mutant larvae. In this case a significant decrease in the amount of the transcripts is evident for four of the six analyzed genes.
    Figure Legend Snippet: Genes corresponding to transcript targets of HP1a in S2 cells appear down-regulated also in DDP1 and PEP mutants larvae. (A) Quantitative RT–PCR analysis of the expression in wild type Ore-R and DDP1 mutant larvae showing a significant decrease in the amount of the transcripts corresponding to all genes. (B) Quantitative RT–PCR analysis in wild type Ore-R and PEP mutant larvae. In this case a significant decrease in the amount of the transcripts is evident for four of the six analyzed genes.

    Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis

    HP1a seems to be involved in forming a complex with hnRNP proteins. (A) Western blot analysis with anti-FLAG antibody of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Molecular weight markers, expressed in kDalton, are reported on the left. (B) Western blot analysis with anti-PEP, anti-DDP1 and anti-HRB87F antibodies of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to nuclear extracts and lysates obtained from 3×10 5 cells before immunopurification (lanes In) are compared with aliquots corresponding to 3×10 6 cells processed for immunopurification (lanes IP HP1 and IP GFP ). Molecular weight markers, expressed in kDalton, are reported on the left. Bands marked with asterisks correspond to mouse IgGs present in the sample and revealed by secondary antibodies. (C) Western blot analysis with anti-FLAG, anti-HRB87F, anti-DDP1 and anti-PEP antibodies of immunopurified fractions obtained from nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a resolved by native-PAGE before (lanes −) and after (lanes +) RNAse treatment. Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Complexes with low-mobility (** and *) and a fast mobility form of HP1a (°) are indicated.
    Figure Legend Snippet: HP1a seems to be involved in forming a complex with hnRNP proteins. (A) Western blot analysis with anti-FLAG antibody of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Molecular weight markers, expressed in kDalton, are reported on the left. (B) Western blot analysis with anti-PEP, anti-DDP1 and anti-HRB87F antibodies of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to nuclear extracts and lysates obtained from 3×10 5 cells before immunopurification (lanes In) are compared with aliquots corresponding to 3×10 6 cells processed for immunopurification (lanes IP HP1 and IP GFP ). Molecular weight markers, expressed in kDalton, are reported on the left. Bands marked with asterisks correspond to mouse IgGs present in the sample and revealed by secondary antibodies. (C) Western blot analysis with anti-FLAG, anti-HRB87F, anti-DDP1 and anti-PEP antibodies of immunopurified fractions obtained from nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a resolved by native-PAGE before (lanes −) and after (lanes +) RNAse treatment. Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Complexes with low-mobility (** and *) and a fast mobility form of HP1a (°) are indicated.

    Techniques Used: Western Blot, Immu-Puri, Molecular Weight, Clear Native PAGE

    The HP1a target transcripts are less stable in cells lacking HP1a. Quantitative RT–PCR analysis of three HP1a target transcripts at different times after blockage of transcription by actinomicyn D treatment. The blue lines and the red lines respectively indicate the transcripts amount in S2 control cells and S2 cells interfered with HP1a dsRNA. Target transcripts correspond to genes: (A) CG5389, (B) CG6779, (C) CG7975. Note that at 120 minutes, the HP1a target transcripts are not detectable in HP1a depleted cells while in control cells their amount is only slightly decreased.
    Figure Legend Snippet: The HP1a target transcripts are less stable in cells lacking HP1a. Quantitative RT–PCR analysis of three HP1a target transcripts at different times after blockage of transcription by actinomicyn D treatment. The blue lines and the red lines respectively indicate the transcripts amount in S2 control cells and S2 cells interfered with HP1a dsRNA. Target transcripts correspond to genes: (A) CG5389, (B) CG6779, (C) CG7975. Note that at 120 minutes, the HP1a target transcripts are not detectable in HP1a depleted cells while in control cells their amount is only slightly decreased.

    Techniques Used: Quantitative RT-PCR

    HP1a can directly bind RNA in vitro and in vivo and interacts with Pol II. (A) Immunolocalization of HP1a and active Pol II on polytene chromosomes of D. melanogaster . Signals produced by the two antibodies show an extensive colocalization. (B) Coimmunoprecipitation of HP1a and Pol II by an anti-HP1a antibody. To test the specificity of HP1a with Pol II interaction, we also probed with an antibody against α-actin. (C) UV crosslinking after incubation of four different concentrations of HP1a with Hsp70 RNA in vitro . Note that radioactive HP1a bands with molecular weights corresponding to an HP1a dimer and monomer are present in the third and fourth lanes. (D) Primer extension of RNA immunoprecipitated from S2 cells with CIA9 antibody. Two signals are present only in HP1a immunoprecipitates of heat-shocked cells (HS). (E) Electromobility shift assay. Left, a diagram of the HP1a fragments used in the gel shift assay. Right, the results of EMSA of radiolabelled HSP70 RNA using the different HP1a fragments (50 ng). The absence of shift (lane d) using the HP1a fragment lacking the chromo-domain strongly suggests that this part of the protein is responsible for the binding of HP1a to the RNA transcripts.
    Figure Legend Snippet: HP1a can directly bind RNA in vitro and in vivo and interacts with Pol II. (A) Immunolocalization of HP1a and active Pol II on polytene chromosomes of D. melanogaster . Signals produced by the two antibodies show an extensive colocalization. (B) Coimmunoprecipitation of HP1a and Pol II by an anti-HP1a antibody. To test the specificity of HP1a with Pol II interaction, we also probed with an antibody against α-actin. (C) UV crosslinking after incubation of four different concentrations of HP1a with Hsp70 RNA in vitro . Note that radioactive HP1a bands with molecular weights corresponding to an HP1a dimer and monomer are present in the third and fourth lanes. (D) Primer extension of RNA immunoprecipitated from S2 cells with CIA9 antibody. Two signals are present only in HP1a immunoprecipitates of heat-shocked cells (HS). (E) Electromobility shift assay. Left, a diagram of the HP1a fragments used in the gel shift assay. Right, the results of EMSA of radiolabelled HSP70 RNA using the different HP1a fragments (50 ng). The absence of shift (lane d) using the HP1a fragment lacking the chromo-domain strongly suggests that this part of the protein is responsible for the binding of HP1a to the RNA transcripts.

    Techniques Used: In Vitro, In Vivo, Produced, Incubation, Immunoprecipitation, Electro Mobility Shift Assay, Electrophoretic Mobility Shift Assay, Binding Assay

    18) Product Images from "A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity"

    Article Title: A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.598492

    Ed-induced polarity as a minimal reconstitution system to model Drosophila neural stem cell spindle orientation. (A) Drosophila neural stem cells (neuroblasts) establish apical-basal polarity in the early stages of mitosis. Both apical and basal polarity complexes (blue and red, respectively) consist of numerous components connected by a complex network of protein-protein interactions and regulatory relationships. At metaphase, the mitotic spindle aligns along this polarity axis through the activity of the apical Pins/Mud/Dlg spindle orientation complex (green). Mitosis proceeds through an asymmetric cell division that is essential for generating differentiated progeny (via the ganglion mother cell, GMC) while also maintaining the stem cell pool through self-renewal. (B) Illustration of how the Ed-induced polarity assay can model Pins-mediated spindle orientation in a minimal reconstituted system (i.e., “X” would represent Pins in this case). Isolated S2 cells initially express an Ed:GFP-X recombinant protein uniformly around the entire cell membrane. Shaking causes collisions that generate cell adhesions wherein cortical Ed:GFP-X proteins concentrate at sites of cell-cell contact within small clusters, the simplest of which is two adhered cells as shown. As cells enter and proceed through mitosis, spindle orientation can be measured relative to the Ed:GFP-X induced crescent similar to how one would with the native Pins crescent in the neuroblast. Note the simplification of this S2 cell system as compared with the complex environment established natively within NBs.
    Figure Legend Snippet: Ed-induced polarity as a minimal reconstitution system to model Drosophila neural stem cell spindle orientation. (A) Drosophila neural stem cells (neuroblasts) establish apical-basal polarity in the early stages of mitosis. Both apical and basal polarity complexes (blue and red, respectively) consist of numerous components connected by a complex network of protein-protein interactions and regulatory relationships. At metaphase, the mitotic spindle aligns along this polarity axis through the activity of the apical Pins/Mud/Dlg spindle orientation complex (green). Mitosis proceeds through an asymmetric cell division that is essential for generating differentiated progeny (via the ganglion mother cell, GMC) while also maintaining the stem cell pool through self-renewal. (B) Illustration of how the Ed-induced polarity assay can model Pins-mediated spindle orientation in a minimal reconstituted system (i.e., “X” would represent Pins in this case). Isolated S2 cells initially express an Ed:GFP-X recombinant protein uniformly around the entire cell membrane. Shaking causes collisions that generate cell adhesions wherein cortical Ed:GFP-X proteins concentrate at sites of cell-cell contact within small clusters, the simplest of which is two adhered cells as shown. As cells enter and proceed through mitosis, spindle orientation can be measured relative to the Ed:GFP-X induced crescent similar to how one would with the native Pins crescent in the neuroblast. Note the simplification of this S2 cell system as compared with the complex environment established natively within NBs.

    Techniques Used: Activity Assay, Isolation, Recombinant

    19) Product Images from "A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection"

    Article Title: A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

    Journal:

    doi: 10.1016/j.freeradbiomed.2005.10.059

    (A) Anti-DmPrx-4783 antiserum recognizes DmPrx-4783 and AsPrx-4783. Crude protein lysates (10 μg) from D. melanogaster S2 cells or from A. stephensi MSQ43 cells were subjected to 12% SDS–PAGE under reducing conditions (DTT), transferred
    Figure Legend Snippet: (A) Anti-DmPrx-4783 antiserum recognizes DmPrx-4783 and AsPrx-4783. Crude protein lysates (10 μg) from D. melanogaster S2 cells or from A. stephensi MSQ43 cells were subjected to 12% SDS–PAGE under reducing conditions (DTT), transferred

    Techniques Used: SDS Page

    Overexpressed AsPrx-4783 protects transfected D. melanogaster S2 cells from RNOS-specific cytotoxicity. D. melanogaster S2 cells were mock transfected (control cells) or transfected with the pTLP55 plasmid encoding AsPrx-4783 (PRX cells). Protection due
    Figure Legend Snippet: Overexpressed AsPrx-4783 protects transfected D. melanogaster S2 cells from RNOS-specific cytotoxicity. D. melanogaster S2 cells were mock transfected (control cells) or transfected with the pTLP55 plasmid encoding AsPrx-4783 (PRX cells). Protection due

    Techniques Used: Transfection, Plasmid Preparation

    20) Product Images from "CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms"

    Article Title: CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2020.10.061

    TIM(S1404) Phosphorylation Promotes TIM Nuclear Retention by Compromising TIM-XPO1 Interaction (A) S1404 is located next to a putative TIM NES : L1394-V1403. S1404 is underlined and shown in gray. Classical NES sequence motif is previously investigated. 58 Φ is hydrophobic amino acid (in gray): Leu; Val; Ile; Phe; or Met. X is any amino acid. (B) Western blots showing reciprocal coimmunoprecipitations (coIPs) to examine the interactions of TIM(WT) or TIM(S1404A) to XPO1 in Drosophila S2 cells expressing pAc- xpo1 -3XFLAG-6XHIS and pAc- per -V5 in the presence or absence of pAc-HA plasmids expressing tim variants. Protein extracts were directly analyzed by immunoblotting (α-V5 for PER) or immunoprecipitated with α-HA or α-FLAG resins to detect baits and interactors. (C and D) Bar graphs displaying quantification of reciprocal coIPs. Values for binding are normalized to amount of bait detected in the IPs and expressed as relative signal intensity (high value = 1). Error bars indicate ± SEM (n = 4); ***p
    Figure Legend Snippet: TIM(S1404) Phosphorylation Promotes TIM Nuclear Retention by Compromising TIM-XPO1 Interaction (A) S1404 is located next to a putative TIM NES : L1394-V1403. S1404 is underlined and shown in gray. Classical NES sequence motif is previously investigated. 58 Φ is hydrophobic amino acid (in gray): Leu; Val; Ile; Phe; or Met. X is any amino acid. (B) Western blots showing reciprocal coimmunoprecipitations (coIPs) to examine the interactions of TIM(WT) or TIM(S1404A) to XPO1 in Drosophila S2 cells expressing pAc- xpo1 -3XFLAG-6XHIS and pAc- per -V5 in the presence or absence of pAc-HA plasmids expressing tim variants. Protein extracts were directly analyzed by immunoblotting (α-V5 for PER) or immunoprecipitated with α-HA or α-FLAG resins to detect baits and interactors. (C and D) Bar graphs displaying quantification of reciprocal coIPs. Values for binding are normalized to amount of bait detected in the IPs and expressed as relative signal intensity (high value = 1). Error bars indicate ± SEM (n = 4); ***p

    Techniques Used: Sequencing, Western Blot, Expressing, Immunoprecipitation, Binding Assay

    CK2 Phosphorylates TIM(S1404) (A) CK2 consensus motifs generated by KinasePhos 2.0. S1404 corresponds to phosphoserine at amino acid position 0 (Support vector machine score = 0.9581). (B) Drosophila S2 cells were transfected with pAc- tim (WT)-HA or pAc- tim (S1404A)-HA and co-transfected with an empty plasmid (pMT-V5-His), pMT- ck2α -V5, or pMT- ck2α (M161K E165D)-V5, referred to as ck2α ( tik ). Protein extracts were incubated with α-HA resin. Total TIM isoforms, TIM(pS1404), and CK2α protein levels were analyzed by western blotting with indicated antibodies. (C) Bar graph showing relative TIM pS1404 levels in (B) normalized to total TIM isoforms. Error bars indicate ± SEM (n = 2); **p
    Figure Legend Snippet: CK2 Phosphorylates TIM(S1404) (A) CK2 consensus motifs generated by KinasePhos 2.0. S1404 corresponds to phosphoserine at amino acid position 0 (Support vector machine score = 0.9581). (B) Drosophila S2 cells were transfected with pAc- tim (WT)-HA or pAc- tim (S1404A)-HA and co-transfected with an empty plasmid (pMT-V5-His), pMT- ck2α -V5, or pMT- ck2α (M161K E165D)-V5, referred to as ck2α ( tik ). Protein extracts were incubated with α-HA resin. Total TIM isoforms, TIM(pS1404), and CK2α protein levels were analyzed by western blotting with indicated antibodies. (C) Bar graph showing relative TIM pS1404 levels in (B) normalized to total TIM isoforms. Error bars indicate ± SEM (n = 2); **p

    Techniques Used: Generated, Plasmid Preparation, Transfection, Incubation, Western Blot

    21) Product Images from "A Novel Pathway of Cell Death in Response to Cytosolic DNA in Drosophila Cells"

    Article Title: A Novel Pathway of Cell Death in Response to Cytosolic DNA in Drosophila Cells

    Journal: Journal of Innate Immunity

    doi: 10.1159/000368276

    Transfected DNA rapidly kills Drosophila S2 cells. The viability of S2 cells was measured by MTT cleavage at 1 h after electroporation, unless otherwise stated. a DNA-dependent cell death of S2 cells after electroporation with 10 µg of CT DNA. The bars represent data from 40 experiments (mean ± SEM) normalised to the no-DNA sample for each experiment. **** p
    Figure Legend Snippet: Transfected DNA rapidly kills Drosophila S2 cells. The viability of S2 cells was measured by MTT cleavage at 1 h after electroporation, unless otherwise stated. a DNA-dependent cell death of S2 cells after electroporation with 10 µg of CT DNA. The bars represent data from 40 experiments (mean ± SEM) normalised to the no-DNA sample for each experiment. **** p

    Techniques Used: Transfection, MTT Assay, Electroporation

    Response of S2 cells and BMMs to different types of DNA. a Cytotoxic effect of DNA from different sources on S2 cells; 10 µg of CT DNA or DNA from salmon sperm, S2 cells, E. coli and pBS plasmid were used in electroporations. Data are from 2 experiments (mean ± range). b Cytotoxic effect of DNA on S2 cells is not dependent on methylation. Cells were electroporated with 10 µg of CT DNA or pBS DNA, either methylated or unmethylated on CpG sequences. Each dot represents a separate electroporated sample, with different symbols denoting 2 different experiments. Response of BMM ( c ) and S2 cells ( d ) to boiled DNA; 10 µg of CT DNA was left intact or boiled for 10 min, transferred to ice and immediately used for electroporation. The bars represent data from 3 ( c ) and 5 ( d ) experiments (mean ± SEM), normalised to the no-DNA sample (* p = 0.029). e Cytotoxic effect of synthetic ssDNA and dsDNA in BMM; 5 µg of CT DNA or synthetic DNA [poly(dA), poly(dT) or poly(dA:dT)] or 1 µg of poly(dA:dT) was electroporated. f Cytotoxic effect of synthetic DNAs in S2 cells, as per panel e. Each dot represents a separate electroporated sample, with the different symbols denoting the 3 different experiments (n = 3, ## p = 0.054). g Absence of cytotoxic effect of poly(dC) on S2 cells; 5 µg of CT DNA or synthetic DNA was used for electroporation. Each dot represents a separate electroporated sample, with the different symbols denoting the different experiments. h Toxic effect of synthetic mixed-base DNA with or without thymidine [poly(dN) and poly(dGCA)] on S2 cells; 10 μg of DNA was used for electroporation. Data are from 5 experiments [CT DNA and poly(dN), mean ± SEM; ** p = 0.0085], or 2 experiments [poly(dGCA), mean ± range].
    Figure Legend Snippet: Response of S2 cells and BMMs to different types of DNA. a Cytotoxic effect of DNA from different sources on S2 cells; 10 µg of CT DNA or DNA from salmon sperm, S2 cells, E. coli and pBS plasmid were used in electroporations. Data are from 2 experiments (mean ± range). b Cytotoxic effect of DNA on S2 cells is not dependent on methylation. Cells were electroporated with 10 µg of CT DNA or pBS DNA, either methylated or unmethylated on CpG sequences. Each dot represents a separate electroporated sample, with different symbols denoting 2 different experiments. Response of BMM ( c ) and S2 cells ( d ) to boiled DNA; 10 µg of CT DNA was left intact or boiled for 10 min, transferred to ice and immediately used for electroporation. The bars represent data from 3 ( c ) and 5 ( d ) experiments (mean ± SEM), normalised to the no-DNA sample (* p = 0.029). e Cytotoxic effect of synthetic ssDNA and dsDNA in BMM; 5 µg of CT DNA or synthetic DNA [poly(dA), poly(dT) or poly(dA:dT)] or 1 µg of poly(dA:dT) was electroporated. f Cytotoxic effect of synthetic DNAs in S2 cells, as per panel e. Each dot represents a separate electroporated sample, with the different symbols denoting the 3 different experiments (n = 3, ## p = 0.054). g Absence of cytotoxic effect of poly(dC) on S2 cells; 5 µg of CT DNA or synthetic DNA was used for electroporation. Each dot represents a separate electroporated sample, with the different symbols denoting the different experiments. h Toxic effect of synthetic mixed-base DNA with or without thymidine [poly(dN) and poly(dGCA)] on S2 cells; 10 μg of DNA was used for electroporation. Data are from 5 experiments [CT DNA and poly(dN), mean ± SEM; ** p = 0.0085], or 2 experiments [poly(dGCA), mean ± range].

    Techniques Used: Plasmid Preparation, Methylation, Electroporation

    Cyclic dinucleotides have no toxic effect on S2 cells, and are not likely to be second messengers in the cell death response to DNA. a Electroporation of S2 cells with CT DNA and cyclic dinucleotides. Cells were electroporated with 10 µg of CT DNA or 20 µg of cyclic di-GMP or cyclic di-AMP, and MTT cleavage was measured 1 h after treatment. Results show the mean and range of duplicate electroporations. b Transfection of S2 cells with CT DNA and cyclic dinucleotides using Lipofectamine 2000; 2 µg of CT DNA and 4 µg of cyclic dinucleotides were transfected into S2 cells complexed with 4 µl of Lipofectamine 2000, and MTT cleavage was measured 18 h after transfection. The bars show data from 4 experiments (mean ± SEM). c Induction of IFN-β mRNA in BMM either untreated (no ZAP) or 2 h after electroporation with either no addition, CT DNA or various amounts of cyclic di-GMP. The real time PCR results shown are mean and range of duplicate assays of IFN-β mRNA relative to HPRT.
    Figure Legend Snippet: Cyclic dinucleotides have no toxic effect on S2 cells, and are not likely to be second messengers in the cell death response to DNA. a Electroporation of S2 cells with CT DNA and cyclic dinucleotides. Cells were electroporated with 10 µg of CT DNA or 20 µg of cyclic di-GMP or cyclic di-AMP, and MTT cleavage was measured 1 h after treatment. Results show the mean and range of duplicate electroporations. b Transfection of S2 cells with CT DNA and cyclic dinucleotides using Lipofectamine 2000; 2 µg of CT DNA and 4 µg of cyclic dinucleotides were transfected into S2 cells complexed with 4 µl of Lipofectamine 2000, and MTT cleavage was measured 18 h after transfection. The bars show data from 4 experiments (mean ± SEM). c Induction of IFN-β mRNA in BMM either untreated (no ZAP) or 2 h after electroporation with either no addition, CT DNA or various amounts of cyclic di-GMP. The real time PCR results shown are mean and range of duplicate assays of IFN-β mRNA relative to HPRT.

    Techniques Used: Electroporation, MTT Assay, Transfection, Real-time Polymerase Chain Reaction

    22) Product Images from "Abelson kinase’s intrinsically disordered region plays essential roles in protein function and protein stability"

    Article Title: Abelson kinase’s intrinsically disordered region plays essential roles in protein function and protein stability

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-020-00703-w

    Abl∆IDR protein accumulates at much higher levels than wildtype Abl. a Immunoblot of 0–6 h embryonic extracts, blotted with antibody to GFP to detect our transgenic proteins. Tubulin serves as a loading control. Despite the fact that both transgenes are driven by the same endogenous abl promotor and the transgenes are at the same chromosomal location, Abl∆IDR protein accumulates at much higher levels than wildtype Abl. b Quantification of mean protein levels from four immunoblots, normalized to both wildtype Abl:GFP and using the loading controls. Colored dots indicate values of the individual blots (Values: 8.2, 11.4, 12.3, and 15.2, Mean: 11.7; Red dot indicates blot shown in a ). Error bar = standard error of the mean. c Immunoblot of extracts of Drosophila S2 cells expressing transgenes encoding wildtype Abl:GFP or Abl∆IDR (at similar transfection efficiencies (see Methods)), both under control of the metallothionine promotor, blotted with antibody to GFP to detect our transgenic proteins. d Representative images of transfected S2 cells stained to visualize F-actin and our transgenic Abl proteins. Wildtype Abl:GFP is enriched in the lamellipodium (arrowhead; highlighted by F-actin) and excluded from nuclei (arrow), while Abl∆IDR:GFP is not enriched in the lamellipodium or excluded from nuclei. Cells expressing Abl∆CR1:GFP (bottom row) resemble those expressing Abl:GFP (enriched in the lamellipodium (arrowhead), excluded from nuclei (arrow)). Scale Bar = 10 µm
    Figure Legend Snippet: Abl∆IDR protein accumulates at much higher levels than wildtype Abl. a Immunoblot of 0–6 h embryonic extracts, blotted with antibody to GFP to detect our transgenic proteins. Tubulin serves as a loading control. Despite the fact that both transgenes are driven by the same endogenous abl promotor and the transgenes are at the same chromosomal location, Abl∆IDR protein accumulates at much higher levels than wildtype Abl. b Quantification of mean protein levels from four immunoblots, normalized to both wildtype Abl:GFP and using the loading controls. Colored dots indicate values of the individual blots (Values: 8.2, 11.4, 12.3, and 15.2, Mean: 11.7; Red dot indicates blot shown in a ). Error bar = standard error of the mean. c Immunoblot of extracts of Drosophila S2 cells expressing transgenes encoding wildtype Abl:GFP or Abl∆IDR (at similar transfection efficiencies (see Methods)), both under control of the metallothionine promotor, blotted with antibody to GFP to detect our transgenic proteins. d Representative images of transfected S2 cells stained to visualize F-actin and our transgenic Abl proteins. Wildtype Abl:GFP is enriched in the lamellipodium (arrowhead; highlighted by F-actin) and excluded from nuclei (arrow), while Abl∆IDR:GFP is not enriched in the lamellipodium or excluded from nuclei. Cells expressing Abl∆CR1:GFP (bottom row) resemble those expressing Abl:GFP (enriched in the lamellipodium (arrowhead), excluded from nuclei (arrow)). Scale Bar = 10 µm

    Techniques Used: Transgenic Assay, Western Blot, Expressing, Transfection, Staining

    23) Product Images from "Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila"

    Article Title: Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006723

    Shams knockdown does not alter the inhibitory effect of cis -Delta on cell aggregation mediated by Notch and trans -Delta. (A) Co-expression of Delta and Notch in a 1:1 ratio in S2 cells significantly decreases their ability to aggregate with S2-Dl cells. Shams KD does not affect the ability of Notch to respond to cis -Delta. (B) Quantification of number of cell aggregates greater than 6 cells after 5 minutes of co-culture. Error bars indicate standard error. * P
    Figure Legend Snippet: Shams knockdown does not alter the inhibitory effect of cis -Delta on cell aggregation mediated by Notch and trans -Delta. (A) Co-expression of Delta and Notch in a 1:1 ratio in S2 cells significantly decreases their ability to aggregate with S2-Dl cells. Shams KD does not affect the ability of Notch to respond to cis -Delta. (B) Quantification of number of cell aggregates greater than 6 cells after 5 minutes of co-culture. Error bars indicate standard error. * P

    Techniques Used: Expressing, Co-Culture Assay

    Shams knockdown does not alter the inhibitory effect of cis -Serrate on cell aggregation mediated by Notch and trans -Delta. (A) Co-expression of Serrate and Notch in a 1:1 ratio in S2 cells significantly decreases their ability to aggregate with S2-Dl cells. Shams KD does not affect the ability of Notch to respond to cis -Serrate. (B) Quantification of number of cell aggregates greater than 6 cells after 5 minutes of co-culture. Error bars indicate standard error. * P
    Figure Legend Snippet: Shams knockdown does not alter the inhibitory effect of cis -Serrate on cell aggregation mediated by Notch and trans -Delta. (A) Co-expression of Serrate and Notch in a 1:1 ratio in S2 cells significantly decreases their ability to aggregate with S2-Dl cells. Shams KD does not affect the ability of Notch to respond to cis -Serrate. (B) Quantification of number of cell aggregates greater than 6 cells after 5 minutes of co-culture. Error bars indicate standard error. * P

    Techniques Used: Expressing, Co-Culture Assay

    24) Product Images from "Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals"

    Article Title: Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014303

    Syx5 suppression in Drosophila S2 cells decreases copper accumulation. Metal accumulation was measured by ICP-AES in control (black) and Syx5 (grey) RNAi suppression cells grown in basal media (A). Syx5 gene expression was suppressed to 19–36% of wild-type levels. Values are mean with s.e.m of eight replicates over two experiments, normalized against control cells. Mean copper accumulation during a 24 h exposure to 2 µM Cu was measured using 64 Cu in S2 cell lines stably over-expressing Ctr1A , Ctr1B or an empty vector control and normalized to total cellular protein (B). Error bars are s.e.m. from nine replicates over three experiments. Syx5 gene expression levels relative to wild-type were 18–41% (control cells), 25-41% (Ctr1A) and 23-31% (Ctr1B). Copper accumulation is reduced by Syx5 suppression (A) even when Ctr1A or Ctr1B is over-expressed (B): suppression of Syx5 reduces copper levels to 50–70% of wild-type in all cell lines. *Significant difference between control and Syx5 suppression cells, determined by an independent samples T-Test (P
    Figure Legend Snippet: Syx5 suppression in Drosophila S2 cells decreases copper accumulation. Metal accumulation was measured by ICP-AES in control (black) and Syx5 (grey) RNAi suppression cells grown in basal media (A). Syx5 gene expression was suppressed to 19–36% of wild-type levels. Values are mean with s.e.m of eight replicates over two experiments, normalized against control cells. Mean copper accumulation during a 24 h exposure to 2 µM Cu was measured using 64 Cu in S2 cell lines stably over-expressing Ctr1A , Ctr1B or an empty vector control and normalized to total cellular protein (B). Error bars are s.e.m. from nine replicates over three experiments. Syx5 gene expression levels relative to wild-type were 18–41% (control cells), 25-41% (Ctr1A) and 23-31% (Ctr1B). Copper accumulation is reduced by Syx5 suppression (A) even when Ctr1A or Ctr1B is over-expressed (B): suppression of Syx5 reduces copper levels to 50–70% of wild-type in all cell lines. *Significant difference between control and Syx5 suppression cells, determined by an independent samples T-Test (P

    Techniques Used: Expressing, Stable Transfection, Plasmid Preparation

    25) Product Images from "The H3K4 Demethylase Lid Associates with and Inhibits Histone Deacetylase Rpd3 ▿"

    Article Title: The H3K4 Demethylase Lid Associates with and Inhibits Histone Deacetylase Rpd3 ▿

    Journal:

    doi: 10.1128/MCB.01643-08

    Lid inhibits the HDAC activity of Rpd3 in vivo. (A) Identification of Rpd3 targets in S2 cells. S2 cells were treated with 100 ng/ml TSA for 48 h to repress deacetylation by Rpd3. RNA was isolated from dimethyl sulfoxide-treated control cells (ctrl) or
    Figure Legend Snippet: Lid inhibits the HDAC activity of Rpd3 in vivo. (A) Identification of Rpd3 targets in S2 cells. S2 cells were treated with 100 ng/ml TSA for 48 h to repress deacetylation by Rpd3. RNA was isolated from dimethyl sulfoxide-treated control cells (ctrl) or

    Techniques Used: Activity Assay, In Vivo, Isolation

    26) Product Images from "Interplay of pericentromeric genome organization and chromatin landscape regulates the expression of Drosophila melanogaster heterochromatic genes"

    Article Title: Interplay of pericentromeric genome organization and chromatin landscape regulates the expression of Drosophila melanogaster heterochromatic genes

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-020-00358-4

    Characteristics of the pericentromeric Het TADs  a  comparative heatmap for the enrichment of various genomic and epigenomic features across scaled average Het TAD borders (30 kb) and intra-TAD regions (150 kb). TAD borders are enriched in MARs and BEAF32 while other architectural proteins are present predominantly at the intra-TAD interactions (for the  p  values of the overlap of features with TAD borders, see Additional file   1 : Figure S5 and S6). Active histone modifications, H3K4me1, H3K4me3, H3K9ac are present within the TADs, whereas H3K36me3 and heterochromatic marks—H3K9me2/3 and HP1a—are present both at the intra-TAD and TAD border regions.  b  Representative snapshot showing the overlap of Het TAD organization in chr 2L with the published replication timing domains in S2 cells—late-replicating regions coincide with inactive Het TADs while active Het TADs have both early- and late-replicating regions within them
    Figure Legend Snippet: Characteristics of the pericentromeric Het TADs a comparative heatmap for the enrichment of various genomic and epigenomic features across scaled average Het TAD borders (30 kb) and intra-TAD regions (150 kb). TAD borders are enriched in MARs and BEAF32 while other architectural proteins are present predominantly at the intra-TAD interactions (for the p values of the overlap of features with TAD borders, see Additional file 1 : Figure S5 and S6). Active histone modifications, H3K4me1, H3K4me3, H3K9ac are present within the TADs, whereas H3K36me3 and heterochromatic marks—H3K9me2/3 and HP1a—are present both at the intra-TAD and TAD border regions. b Representative snapshot showing the overlap of Het TAD organization in chr 2L with the published replication timing domains in S2 cells—late-replicating regions coincide with inactive Het TADs while active Het TADs have both early- and late-replicating regions within them

    Techniques Used:

    27) Product Images from "Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway"

    Article Title: Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

    Journal:

    doi: 10.1038/sj.emboj.7600807

    Iap2 and TAB are located below Imd in the Imd signaling cascade. ( A ) Iap2 and TAB are located downstream of Imd. Overexpression of Relish constructs or Imd caused an activation of  Att  response in S2 cells.  Att  induction caused by expression of either
    Figure Legend Snippet: Iap2 and TAB are located below Imd in the Imd signaling cascade. ( A ) Iap2 and TAB are located downstream of Imd. Overexpression of Relish constructs or Imd caused an activation of Att response in S2 cells. Att induction caused by expression of either

    Techniques Used: Radial Immuno Diffusion, Over Expression, Construct, Activation Assay, Expressing

    Targeted RNAi of Iap2 or TAB reduces the Imd pathway activity. ( A ) The effect of targeted Iap2 or TAB RNAi on Imd and Toll pathways. S2 cells were transfected either with Att -luc (the Imd pathway) or with Drs -luc reporter plasmid together with Toll10b
    Figure Legend Snippet: Targeted RNAi of Iap2 or TAB reduces the Imd pathway activity. ( A ) The effect of targeted Iap2 or TAB RNAi on Imd and Toll pathways. S2 cells were transfected either with Att -luc (the Imd pathway) or with Drs -luc reporter plasmid together with Toll10b

    Techniques Used: Radial Immuno Diffusion, Activity Assay, Transfection, Plasmid Preparation

    RNAi analysis of gene products regulating the expression of Attacin via the Imd pathway in S2 cells
    Figure Legend Snippet: RNAi analysis of gene products regulating the expression of Attacin via the Imd pathway in S2 cells

    Techniques Used: Expressing, Radial Immuno Diffusion

    Schematic representation of the Imd signaling pathway in  Drosophila  S2 cells. The asterisk ( * ). Two asterisks ( ** ) represents
    Figure Legend Snippet: Schematic representation of the Imd signaling pathway in Drosophila S2 cells. The asterisk ( * ). Two asterisks ( ** ) represents

    Techniques Used: Radial Immuno Diffusion

    RNAi-based screen to identify components of the Imd signaling cascade in S2 cells. ( A ) RNAi-mediated gene silencing effectively and specifically decreases the mRNA level of the targeted gene in S2 cells. A total of 2.5 × 10 6 S2 cells in six-well
    Figure Legend Snippet: RNAi-based screen to identify components of the Imd signaling cascade in S2 cells. ( A ) RNAi-mediated gene silencing effectively and specifically decreases the mRNA level of the targeted gene in S2 cells. A total of 2.5 × 10 6 S2 cells in six-well

    Techniques Used: Radial Immuno Diffusion

    28) Product Images from "CLOCK stabilizes CYCLE to initiate clock function in Drosophila"

    Article Title: CLOCK stabilizes CYCLE to initiate clock function in Drosophila

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

    doi: 10.1073/pnas.1707143114

    CYC protein is stabilized when coexpressed with CLK. S2 cells transfected with pMK- cyc -FLAG ( cyc -Fg) plasmid alone or in combination with pAc- Clk -V5 ( Clk -V5) plasmid were incubated with CuSO 4 for 1 h to induce cyc -Fg expression and then treated with CHX to inhibit translation. ( A ) S2 cells transfected with cyc -Fg. ( B ) S2 cells transfected with cyc -Fg and treated with MG132 at 0 h. ( C ) S2 cells cotransfected with cyc -Fg and Clk -V5. Proteins were extracted from cells harvested at the indicated times after CHX addition and used to prepare Western blots that were probed with anti-FLAG, anti-CLK, and anti–β-ACTIN antibodies. ( D ) and plotted as the mean value ± SEM from four independent experiments. ( E ) Protein extracts from cells transfected with Clk -V5 alone or Clk -V5 and cyc -Fg were subjected to immunoprecipitation using anti-FLAG antibody. Western blots containing cell extracts (Input) or immune complexes (IP) were probed with anti-FLAG and anti-CLK antibodies.
    Figure Legend Snippet: CYC protein is stabilized when coexpressed with CLK. S2 cells transfected with pMK- cyc -FLAG ( cyc -Fg) plasmid alone or in combination with pAc- Clk -V5 ( Clk -V5) plasmid were incubated with CuSO 4 for 1 h to induce cyc -Fg expression and then treated with CHX to inhibit translation. ( A ) S2 cells transfected with cyc -Fg. ( B ) S2 cells transfected with cyc -Fg and treated with MG132 at 0 h. ( C ) S2 cells cotransfected with cyc -Fg and Clk -V5. Proteins were extracted from cells harvested at the indicated times after CHX addition and used to prepare Western blots that were probed with anti-FLAG, anti-CLK, and anti–β-ACTIN antibodies. ( D ) and plotted as the mean value ± SEM from four independent experiments. ( E ) Protein extracts from cells transfected with Clk -V5 alone or Clk -V5 and cyc -Fg were subjected to immunoprecipitation using anti-FLAG antibody. Western blots containing cell extracts (Input) or immune complexes (IP) were probed with anti-FLAG and anti-CLK antibodies.

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot, Immunoprecipitation

    29) Product Images from "Pharmacological Analysis of Drosophila melanogaster ?-Secretase with Respect to Differential Proteolysis of Notch and APP"

    Article Title: Pharmacological Analysis of Drosophila melanogaster ?-Secretase with Respect to Differential Proteolysis of Notch and APP

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.109.062471

    Processing of D. melanogaster APPL. Immunoblot analysis of epitope-tagged APPL expressed in D. melanogaster S2 cells treated with different inhibitor compounds that target either γ-secretase, endosome acidification, or metalloprotease activity (see Materials and Methods ). Lane numbers, expression constructs, and drug combinations are indicated above each immunoblot. A, full-length APPL is processed into CTFs that can be detected more readily by preventing their processing by γ-secretase using the inhibitor CpnE. B, APPL also releases a highly unstable γ-secretase-dependent fragment (AICD) that can be detected in longer exposures. C, extended gel electrophoresis identifies three APPL CTFs (a, b, and c) with CTFc generation requiring metalloprotease activity. ConA, endosome acidification inhibitor Concanamycin A; Epo, proteasome inhibitor epoximicin; GM6001, α-secretase metalloprotease inhibitor. β-Tubulin serves as the loading control in all immunoblots.
    Figure Legend Snippet: Processing of D. melanogaster APPL. Immunoblot analysis of epitope-tagged APPL expressed in D. melanogaster S2 cells treated with different inhibitor compounds that target either γ-secretase, endosome acidification, or metalloprotease activity (see Materials and Methods ). Lane numbers, expression constructs, and drug combinations are indicated above each immunoblot. A, full-length APPL is processed into CTFs that can be detected more readily by preventing their processing by γ-secretase using the inhibitor CpnE. B, APPL also releases a highly unstable γ-secretase-dependent fragment (AICD) that can be detected in longer exposures. C, extended gel electrophoresis identifies three APPL CTFs (a, b, and c) with CTFc generation requiring metalloprotease activity. ConA, endosome acidification inhibitor Concanamycin A; Epo, proteasome inhibitor epoximicin; GM6001, α-secretase metalloprotease inhibitor. β-Tubulin serves as the loading control in all immunoblots.

    Techniques Used: Activity Assay, Expressing, Construct, Nucleic Acid Electrophoresis, Western Blot

    Proteolytic processing of APP and Notch. A, comparison of proteolytic cleavage site locations with respect to the membrane topology of the two γ-secretase substrates Notch and APP. Notch is cleaved extracellularly by a furin-like protease and ADAM metalloprotease, whereas APP is cleaved extracellularly at similar locations by β-site of APP cleaving enzyme (BACE) aspartyl proteases and ADAM metalloproteases. Extracellular cleavages are followed by intramembrane proteolysis performed by γ-secretase for both substrates. B, diagram depicting the human Presenilin domains that are targeted by the four well characterized γ-secretase inhibitor compounds (DBZ, CpnE, DAPT, and DFK167) that we used for dose-response studies of APPL and Notch proteolysis in  D. melanogaster  S2 cells. The mature PS NTF is depicted in green and the CTF is shown in red.
    Figure Legend Snippet: Proteolytic processing of APP and Notch. A, comparison of proteolytic cleavage site locations with respect to the membrane topology of the two γ-secretase substrates Notch and APP. Notch is cleaved extracellularly by a furin-like protease and ADAM metalloprotease, whereas APP is cleaved extracellularly at similar locations by β-site of APP cleaving enzyme (BACE) aspartyl proteases and ADAM metalloproteases. Extracellular cleavages are followed by intramembrane proteolysis performed by γ-secretase for both substrates. B, diagram depicting the human Presenilin domains that are targeted by the four well characterized γ-secretase inhibitor compounds (DBZ, CpnE, DAPT, and DFK167) that we used for dose-response studies of APPL and Notch proteolysis in D. melanogaster S2 cells. The mature PS NTF is depicted in green and the CTF is shown in red.

    Techniques Used:

    Dose-response analysis of Notch and APPL cleavage sensitivity to γ-secretase inhibitors. A, representative examples of immunoblots used for dose-response studies of Notch and APPL with four different γ-secretase inhibitors. The substrate under analysis is shown in the black box in the upper left corner of each immunoblot; the inhibitor drug used is shown at upper left above each immunoblot set, with drug concentrations indicated above each lane. Hypotonic lysates from S2 cells were resolved on 3 to 8% Tris-acetate or 16.5% Tris-Tricine gels and immunoprobed with antibodies to the C termini of the expressed constructs (see Materials and Methods ). Increasing drug concentrations led to a progressive accumulation of APPL CTFs and a progressive decrease in Notch NICD levels. Because of difficulties in detecting the highly unstable APPL AICD, immunoblot quantifications to establish the pharmacological potencies of inhibitors are based on CTFa abundance for the APPL studies, whereas NICD levels were monitored for the Notch dose-response studies. Replicate numbers of dose-response immunoblots performed for each substrate/inhibitor pair were as follows: Notch/DBZ (5), Notch/CpnE (5), Notch/DAPT (7), Notch/DFK167 (7), APPL/DBZ (4), APPL/CpnE (4), APPL/DAPT (4), and APPL/DFK167 (5). B, dose-response curves for Notch and APPL are shown in black and orange, respectively, for the γ-secretase inhibitors DBZ, CpnE, DAPT, and DFK167, as indicated. The tested concentration range for each compound analyzed was as follows: DBZ, 0.1–250 nM; CpnE, 1–1000 nM; DAPT, 25–2500 nM; DFK167, 1–150 μM. C, the estimated IC 50 values show that DAPT and DFK167 exhibit modest selectivity in blocking Notch and APPL intramembrane proteolysis. Notch and APPL IC 50 estimates for DAPT and DFK167 differ statistically ( p
    Figure Legend Snippet: Dose-response analysis of Notch and APPL cleavage sensitivity to γ-secretase inhibitors. A, representative examples of immunoblots used for dose-response studies of Notch and APPL with four different γ-secretase inhibitors. The substrate under analysis is shown in the black box in the upper left corner of each immunoblot; the inhibitor drug used is shown at upper left above each immunoblot set, with drug concentrations indicated above each lane. Hypotonic lysates from S2 cells were resolved on 3 to 8% Tris-acetate or 16.5% Tris-Tricine gels and immunoprobed with antibodies to the C termini of the expressed constructs (see Materials and Methods ). Increasing drug concentrations led to a progressive accumulation of APPL CTFs and a progressive decrease in Notch NICD levels. Because of difficulties in detecting the highly unstable APPL AICD, immunoblot quantifications to establish the pharmacological potencies of inhibitors are based on CTFa abundance for the APPL studies, whereas NICD levels were monitored for the Notch dose-response studies. Replicate numbers of dose-response immunoblots performed for each substrate/inhibitor pair were as follows: Notch/DBZ (5), Notch/CpnE (5), Notch/DAPT (7), Notch/DFK167 (7), APPL/DBZ (4), APPL/CpnE (4), APPL/DAPT (4), and APPL/DFK167 (5). B, dose-response curves for Notch and APPL are shown in black and orange, respectively, for the γ-secretase inhibitors DBZ, CpnE, DAPT, and DFK167, as indicated. The tested concentration range for each compound analyzed was as follows: DBZ, 0.1–250 nM; CpnE, 1–1000 nM; DAPT, 25–2500 nM; DFK167, 1–150 μM. C, the estimated IC 50 values show that DAPT and DFK167 exhibit modest selectivity in blocking Notch and APPL intramembrane proteolysis. Notch and APPL IC 50 estimates for DAPT and DFK167 differ statistically ( p

    Techniques Used: Western Blot, Construct, Concentration Assay, Blocking Assay

    Relationship of metalloprotease and γ-secretase Notch cleavage in D. melanogaster cells. A, the Notch receptor is initially synthesized as an ∼300-kDa precursor that is processed by furin-like enzyme(s) in the trans -Golgi compartment. This cleavage leads to the production of a heterodimeric Notch receptor, which is further processed at the cell surface in a ligand-dependent manner by ADAM and γ-secretase. The ADAM cleavage occurs at an extracellular site and removes the Notch ectodomain, whereas γ-secretase cleavage (GS) occurs at multiple positions of the Notch transmembrane domain, leading to the release of NICD that functions as a nuclear transcriptional regulator. B, immunoblot analysis of Notch biochemical processing after treatment of Notch-expressing S2 cells with pharmacological inhibitors metalloproteases involved in Notch ectodomain shedding (BB94), γ-secretase Notch cleavage (CpnE), and both processes together (BB94 and CpnE). Expression constructs and drug combinations are shown above the immunoblot. The panel at bottom is an enlargement of the fragments detected in the ∼100-kDa range, indicating the membrane-bound fragments produced by the extracellular furin-like and ADAM cleavages, including the putative heterodimeric Notch CTF generated by furin-like cleavage in the trans ).
    Figure Legend Snippet: Relationship of metalloprotease and γ-secretase Notch cleavage in D. melanogaster cells. A, the Notch receptor is initially synthesized as an ∼300-kDa precursor that is processed by furin-like enzyme(s) in the trans -Golgi compartment. This cleavage leads to the production of a heterodimeric Notch receptor, which is further processed at the cell surface in a ligand-dependent manner by ADAM and γ-secretase. The ADAM cleavage occurs at an extracellular site and removes the Notch ectodomain, whereas γ-secretase cleavage (GS) occurs at multiple positions of the Notch transmembrane domain, leading to the release of NICD that functions as a nuclear transcriptional regulator. B, immunoblot analysis of Notch biochemical processing after treatment of Notch-expressing S2 cells with pharmacological inhibitors metalloproteases involved in Notch ectodomain shedding (BB94), γ-secretase Notch cleavage (CpnE), and both processes together (BB94 and CpnE). Expression constructs and drug combinations are shown above the immunoblot. The panel at bottom is an enlargement of the fragments detected in the ∼100-kDa range, indicating the membrane-bound fragments produced by the extracellular furin-like and ADAM cleavages, including the putative heterodimeric Notch CTF generated by furin-like cleavage in the trans ).

    Techniques Used: Synthesized, Expressing, Construct, Produced, Generated

    30) Product Images from "isoTarget: A Genetic Method for Analyzing the Functional Diversity of Splicing Isoforms In Vivo"

    Article Title: isoTarget: A Genetic Method for Analyzing the Functional Diversity of Splicing Isoforms In Vivo

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.108361

    Axonal Enrichment of Wnd Compartmentalizes Wnd-Dscam[TM2] Signaling (A) Hiw suppresses the expression of both Dscam[TM1] and [TM2]. In the brains of third-instar larvae that were trans-heterozygotes of global iso-tagging of Dscam[TM1]::HA and [TM2]::V5, loss of hiw ( hiw ΔN ) elevated the levels of both endogenous Dscam[TM1] and [TM2]. Left: western blots; right: quantification of western blots. Each dot represents the result from one independent experiment. (B) Overexpression of Wnd increases the levels of Dscam[TM2], but not that of [TM1]. Top: the Gal4 4−77 , which is expressed in a small set of PNS neurons and a number of CNS neurons, was used to drive the expression of R recombinase for iso-tagging and the overexpression of Wnd. Each dot represents the result of one independent experiment. (C) Overexpression of Wnd increases endogenous Dscam[TM2] levels in the presynaptic terminals of C4 da neurons. At the third-instar larval stage, Dscam [TM2]::V5 iso-tagging is no longer detectable in C4 da axon terminals. Overexpression of Wnd elevates the level of Dscam[TM2]::V5 iso-tagging in these terminals. (D and E) Wnd promotes Dscam[TM1] and [TM2] expression to a similar extent in cultured S2 cells. S2 cells were transfected with plasmids expressing Wnd and Dscam[TM1]::GFP or [TM2]::GFP with endogenous Dscam 5′ and 3′ UTR. Lysates of S2 cells were blotted by anti-GFP and anti-tubulin antibodies. Each dot represents the result of one independent experiment. (F) Wnd is enriched in axons. GFP-tagged kinase-dead Wnd (GFP::Wnd KD ) was expressed in C4 da neurons by ppk -Gal4. mCD8::RFP was used to label the neurons. Although the GFP::Wnd KD signal is enriched in axon terminals, little is observed in dendrites. Yellow arrows point to major dendritic branches. (G) Subcellular localization expands the functional diversity of splicing isoforms via two different modes. In mode 1, the localization of an isoform in a particular subcellular location (e.g., Dscam[TM1] in dendrites) restrains this isoform from functioning in other compartments. In mode 2, the enrichment of the functional partners (e.g., Wnd and Dock) for a ubiquitously localized isoform (e.g., Dscam[TM2]) leads to isoform-specific subcellular signaling and functions.
    Figure Legend Snippet: Axonal Enrichment of Wnd Compartmentalizes Wnd-Dscam[TM2] Signaling (A) Hiw suppresses the expression of both Dscam[TM1] and [TM2]. In the brains of third-instar larvae that were trans-heterozygotes of global iso-tagging of Dscam[TM1]::HA and [TM2]::V5, loss of hiw ( hiw ΔN ) elevated the levels of both endogenous Dscam[TM1] and [TM2]. Left: western blots; right: quantification of western blots. Each dot represents the result from one independent experiment. (B) Overexpression of Wnd increases the levels of Dscam[TM2], but not that of [TM1]. Top: the Gal4 4−77 , which is expressed in a small set of PNS neurons and a number of CNS neurons, was used to drive the expression of R recombinase for iso-tagging and the overexpression of Wnd. Each dot represents the result of one independent experiment. (C) Overexpression of Wnd increases endogenous Dscam[TM2] levels in the presynaptic terminals of C4 da neurons. At the third-instar larval stage, Dscam [TM2]::V5 iso-tagging is no longer detectable in C4 da axon terminals. Overexpression of Wnd elevates the level of Dscam[TM2]::V5 iso-tagging in these terminals. (D and E) Wnd promotes Dscam[TM1] and [TM2] expression to a similar extent in cultured S2 cells. S2 cells were transfected with plasmids expressing Wnd and Dscam[TM1]::GFP or [TM2]::GFP with endogenous Dscam 5′ and 3′ UTR. Lysates of S2 cells were blotted by anti-GFP and anti-tubulin antibodies. Each dot represents the result of one independent experiment. (F) Wnd is enriched in axons. GFP-tagged kinase-dead Wnd (GFP::Wnd KD ) was expressed in C4 da neurons by ppk -Gal4. mCD8::RFP was used to label the neurons. Although the GFP::Wnd KD signal is enriched in axon terminals, little is observed in dendrites. Yellow arrows point to major dendritic branches. (G) Subcellular localization expands the functional diversity of splicing isoforms via two different modes. In mode 1, the localization of an isoform in a particular subcellular location (e.g., Dscam[TM1] in dendrites) restrains this isoform from functioning in other compartments. In mode 2, the enrichment of the functional partners (e.g., Wnd and Dock) for a ubiquitously localized isoform (e.g., Dscam[TM2]) leads to isoform-specific subcellular signaling and functions.

    Techniques Used: Expressing, Western Blot, Over Expression, Cell Culture, Transfection, Functional Assay

    Dscam[TM1] and [TM2] Exhibit Similar Biochemical Properties and Cellular Functions When They Are Localized in the Same Subcellular Compartment (A and B) Dock preferentially interacts with endogenous Dscam[TM2] in vivo . (A) Brain lysates of larvae with global iso-tagging of [TM1]::HA or [TM2]::V5 were immunoprecipitated by anti-HA or V5 beads, respectively, and immunoblotted with an anti-Dscam antibody that recognizes both [TM1] and [TM2] and an anti-Dock antibody. This experiment was repeated twice. (B) Brain lysates of larvae that expressed [TM1]::GFP or [TM2]::GFP through the endogenous Dscam promoter were immunoprecipitated by an anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-GFP and anti-Dock antibodies. This experiment was repeated three times. (C–F) Dscam[TM2] requires Dock to promote presynaptic terminal growth. Shown are representative images of A4–A6. Overexpressing a Dscam[TM2]::GFP transgene significantly promotes axonal growth in C4 da neurons (C) (D). Although the loss of dock does not affect C4 da axon terminals (E), it completely abolishes the overgrowth caused by Dscam[TM2]::GFP overexpression (F). (G) Quantification of the number of C4 da axon connectives. (H) Dock binds to [TM1] and [TM2] in similar affinity in cultured S2 cells. Lysates of S2 cells expressing mCD8::GFP, Dscam[TM1]::GFP or [TM2]::GFP were immunoprecipitated with an anti-GFP antibody. Inputs and immunoprecipitates were blotted by anti-GFP and anti-Dock antibodies. This experiment was repeated three times. (I–K) Transgenic Dscam[TM1] requires Dock to promote presynaptic terminal growth. Compared with WT (I), overexpressing [TM1]::GFP transgene significantly promotes axonal growth in C4 da neurons (J), which is completely abolished by loss of dock (K). (L) Quantification of the number of C4 da axon connectives from A4–A6. See also Figure S6 .
    Figure Legend Snippet: Dscam[TM1] and [TM2] Exhibit Similar Biochemical Properties and Cellular Functions When They Are Localized in the Same Subcellular Compartment (A and B) Dock preferentially interacts with endogenous Dscam[TM2] in vivo . (A) Brain lysates of larvae with global iso-tagging of [TM1]::HA or [TM2]::V5 were immunoprecipitated by anti-HA or V5 beads, respectively, and immunoblotted with an anti-Dscam antibody that recognizes both [TM1] and [TM2] and an anti-Dock antibody. This experiment was repeated twice. (B) Brain lysates of larvae that expressed [TM1]::GFP or [TM2]::GFP through the endogenous Dscam promoter were immunoprecipitated by an anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-GFP and anti-Dock antibodies. This experiment was repeated three times. (C–F) Dscam[TM2] requires Dock to promote presynaptic terminal growth. Shown are representative images of A4–A6. Overexpressing a Dscam[TM2]::GFP transgene significantly promotes axonal growth in C4 da neurons (C) (D). Although the loss of dock does not affect C4 da axon terminals (E), it completely abolishes the overgrowth caused by Dscam[TM2]::GFP overexpression (F). (G) Quantification of the number of C4 da axon connectives. (H) Dock binds to [TM1] and [TM2] in similar affinity in cultured S2 cells. Lysates of S2 cells expressing mCD8::GFP, Dscam[TM1]::GFP or [TM2]::GFP were immunoprecipitated with an anti-GFP antibody. Inputs and immunoprecipitates were blotted by anti-GFP and anti-Dock antibodies. This experiment was repeated three times. (I–K) Transgenic Dscam[TM1] requires Dock to promote presynaptic terminal growth. Compared with WT (I), overexpressing [TM1]::GFP transgene significantly promotes axonal growth in C4 da neurons (J), which is completely abolished by loss of dock (K). (L) Quantification of the number of C4 da axon connectives from A4–A6. See also Figure S6 .

    Techniques Used: In Vivo, Immunoprecipitation, Over Expression, Cell Culture, Expressing, Transgenic Assay

    31) Product Images from "Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila"

    Article Title: Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila

    Journal: Cell Research

    doi: 10.1038/cr.2014.29

    HIB affects Su(fu) level by modulating Crn cellular localization. (A - C'''') S2 cells transfected with UAS-HA-Crn , UAS-Flag-HIB , or both were immunostained to show the subcellular localization of HA-Crn and Flag-HIB. The nuclei were stained with DAPI (blue) and the membrane was stained with TRITC-labeled phalloidin that preferentially labels filamentous actin (F-actin) (red). When expressed alone, HA-Crn was located mainly in the cytoplasm and barely in the nucleus (A - A''') , whereas Flag-HIB was located in the nucleus (B - B''') . When co-expressed with Flag-HIB, HA-Crn was co-localized with Flag-HIB at speckle-like sites in the nucleus (C - C''') ; however, treating cells with MG132 resulted in cytoplasmic localization of HA-Crn (C'''') . (D - D''') LMB treatment resulted in nuclear localization of HA-Crn in the absence of Flag-HIB co-expression in S2 cells. (E - G''') Wing discs expressing UAS-HA-Crn alone (E - E''') or together with UAS-Flag-HIB (F - F''') or UAS-Hh (G - G''') with MS1096 were immunostained to show HA (green), Su(fu) (red in E''' , F''' ), DAPI (blue) and F-actin (red in E'' , F'' ). High-magnification views of anterior compartment cells were shown in (E - E'') , (F - F'') , and (G - G''') . When expressed alone, HA-Crn was located mainly in cytoplasm (E - E'') and did not affect endogenous Su(fu) level (E''') ; however, when co-overexpressed with Flag-HIB, Crn was localized in the nucleus (F - F'') and downregulated endogenous Su(fu) level (F''') . Similarly, when co-overexpressed with UAS-Hh , Crn was also localized in the nucleus (G - G''') . (H - H''') LMB treatment led to nuclear localization of HA-Crn in the wing discs in the absence of HIB or Hh co-expression. (I) Endogenous Su(fu) level was reduced as shown by western blot in LMB-treated wing discs; however, the endogenous Su(fu) level was restored or even upregulated when crn was knocked down at the same time. (J) Quantification of the western blot results of endogenous Su(fu) level in (I) analyzed by ImageJ. (K) Co-expression of Flag-HIB with HA-Crn decreased Su(fu) protein level in S2 cells as determined by western blot analysis. Actin was used as a loading control.
    Figure Legend Snippet: HIB affects Su(fu) level by modulating Crn cellular localization. (A - C'''') S2 cells transfected with UAS-HA-Crn , UAS-Flag-HIB , or both were immunostained to show the subcellular localization of HA-Crn and Flag-HIB. The nuclei were stained with DAPI (blue) and the membrane was stained with TRITC-labeled phalloidin that preferentially labels filamentous actin (F-actin) (red). When expressed alone, HA-Crn was located mainly in the cytoplasm and barely in the nucleus (A - A''') , whereas Flag-HIB was located in the nucleus (B - B''') . When co-expressed with Flag-HIB, HA-Crn was co-localized with Flag-HIB at speckle-like sites in the nucleus (C - C''') ; however, treating cells with MG132 resulted in cytoplasmic localization of HA-Crn (C'''') . (D - D''') LMB treatment resulted in nuclear localization of HA-Crn in the absence of Flag-HIB co-expression in S2 cells. (E - G''') Wing discs expressing UAS-HA-Crn alone (E - E''') or together with UAS-Flag-HIB (F - F''') or UAS-Hh (G - G''') with MS1096 were immunostained to show HA (green), Su(fu) (red in E''' , F''' ), DAPI (blue) and F-actin (red in E'' , F'' ). High-magnification views of anterior compartment cells were shown in (E - E'') , (F - F'') , and (G - G''') . When expressed alone, HA-Crn was located mainly in cytoplasm (E - E'') and did not affect endogenous Su(fu) level (E''') ; however, when co-overexpressed with Flag-HIB, Crn was localized in the nucleus (F - F'') and downregulated endogenous Su(fu) level (F''') . Similarly, when co-overexpressed with UAS-Hh , Crn was also localized in the nucleus (G - G''') . (H - H''') LMB treatment led to nuclear localization of HA-Crn in the wing discs in the absence of HIB or Hh co-expression. (I) Endogenous Su(fu) level was reduced as shown by western blot in LMB-treated wing discs; however, the endogenous Su(fu) level was restored or even upregulated when crn was knocked down at the same time. (J) Quantification of the western blot results of endogenous Su(fu) level in (I) analyzed by ImageJ. (K) Co-expression of Flag-HIB with HA-Crn decreased Su(fu) protein level in S2 cells as determined by western blot analysis. Actin was used as a loading control.

    Techniques Used: Transfection, Staining, Labeling, Expressing, Western Blot

    32) Product Images from "Mutation of the Drosophila vesicular GABA transporter disrupts visual figure detection"

    Article Title: Mutation of the Drosophila vesicular GABA transporter disrupts visual figure detection

    Journal: The Journal of Experimental Biology

    doi: 10.1242/jeb.036053

    Expression of dVGAT protein. (A) Western blots of head homogenates probed with an antibody to dVGAT show a single major band. Standards (in kDa) are indicated on the left. (B) Cultured Drosophila S2 cells expressing the dVGAT cDNA and labeled with the antibody to dVGAT show punctate intracellular labeling. Scale bar, 10 μm. (C) Glycerol velocity gradients show that dVGAT partially co-sediments with a marker for synaptic vesicles (CSP).
    Figure Legend Snippet: Expression of dVGAT protein. (A) Western blots of head homogenates probed with an antibody to dVGAT show a single major band. Standards (in kDa) are indicated on the left. (B) Cultured Drosophila S2 cells expressing the dVGAT cDNA and labeled with the antibody to dVGAT show punctate intracellular labeling. Scale bar, 10 μm. (C) Glycerol velocity gradients show that dVGAT partially co-sediments with a marker for synaptic vesicles (CSP).

    Techniques Used: Expressing, Western Blot, Cell Culture, Labeling, Marker

    33) Product Images from "TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation"

    Article Title: TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21228871

    Analysis of Trf2 and D1 genes mRNA levels in salivary glands of the third instar larvae, adult flies and S2 cells. The relative expression levels of Trf2 and D1 genes were examined using qRT-PCR as described in materials and methods. ( A ) Expression levels of Trf2 and D1 genes detected in salivary glands of: control UAS-Tris/+ larvae and after Trf2 knock-down by ddRNAi ( sgs > Tris ). ( B ) Expression levels of Trf2 and D1 genes in lawc p1 /EF520 adult flies compared to control EF520/+ flies. ( C ) Expression levels of Trf2 and D1 genes in lawc p1 /Df(1)RA2 adult flies compared to control Df(1)RA2/+ flies; ( D ) expression levels of Trf2 and D1 genes in the S2 cells after RNAi-mediated silencing of Trf2 (S2-TRFi) compared to control (S2 cells treated with dsRNA corresponding to eGFP gene). Data are presented as the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Analysis of Trf2 and D1 genes mRNA levels in salivary glands of the third instar larvae, adult flies and S2 cells. The relative expression levels of Trf2 and D1 genes were examined using qRT-PCR as described in materials and methods. ( A ) Expression levels of Trf2 and D1 genes detected in salivary glands of: control UAS-Tris/+ larvae and after Trf2 knock-down by ddRNAi ( sgs > Tris ). ( B ) Expression levels of Trf2 and D1 genes in lawc p1 /EF520 adult flies compared to control EF520/+ flies. ( C ) Expression levels of Trf2 and D1 genes in lawc p1 /Df(1)RA2 adult flies compared to control Df(1)RA2/+ flies; ( D ) expression levels of Trf2 and D1 genes in the S2 cells after RNAi-mediated silencing of Trf2 (S2-TRFi) compared to control (S2 cells treated with dsRNA corresponding to eGFP gene). Data are presented as the mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Quantitative RT-PCR

    RNAi-mediated depletion of Trf2 leads to micronuclei formation and disturbs nuclear envelope. Confocal immunofluorescence images of Drosophila S2 cells ( A ) before and ( B ) after RNAi-mediated silencing of Trf2 . Cells were stained for SytoxGreen to highlight DNA (green) and anti-Lamin Dm to visualized nuclear envelope (cian). The third column represents merged images. Arrowheads pointe to micronucleus, arrows indicate Lamin negative nuclear envelope. ( C ) Quantification of cells with micronuclei from control (Contr, n = 350) and Trf2 dsRNA transfected cells ( Trf2i , n = 229) from three independent experiments. A p -value from Student’s t -test was 0.0047. Error bars: SD.
    Figure Legend Snippet: RNAi-mediated depletion of Trf2 leads to micronuclei formation and disturbs nuclear envelope. Confocal immunofluorescence images of Drosophila S2 cells ( A ) before and ( B ) after RNAi-mediated silencing of Trf2 . Cells were stained for SytoxGreen to highlight DNA (green) and anti-Lamin Dm to visualized nuclear envelope (cian). The third column represents merged images. Arrowheads pointe to micronucleus, arrows indicate Lamin negative nuclear envelope. ( C ) Quantification of cells with micronuclei from control (Contr, n = 350) and Trf2 dsRNA transfected cells ( Trf2i , n = 229) from three independent experiments. A p -value from Student’s t -test was 0.0047. Error bars: SD.

    Techniques Used: Immunofluorescence, Staining, Transfection

    34) Product Images from "miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting"

    Article Title: miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006257

    Interactions between miRNAs (miR-71 and miR-263) and their targets CHS1 and CHT10 in locusts. (A, B) Luciferase reporter assays were analyzed in S2 cells co-transfected with miR-71 or miR-263 overexpression vectors and psi-CHECK2 vectors containing wild-type (WT) or mutant (MT, the 8 nt of the region that corresponds to the miRNA seed mutated) target gene sequences of CHS1 (A) and CHT10 (B) ( n = 6). (C, D) RIP was performed with an anti-Ago-1 antibody. qPCR analysis was performed to amplify the CHS1 and CHT10 mRNAs from the Ago-1 immunoprecipitates from extracts of integument tissue treated with agomir-71 or agomir-263 48 h later compared with the agomir controls (agomir-NC). The data for the luciferase activities and qPCR analyses are presented as means ± SEM ( n = 6). * p
    Figure Legend Snippet: Interactions between miRNAs (miR-71 and miR-263) and their targets CHS1 and CHT10 in locusts. (A, B) Luciferase reporter assays were analyzed in S2 cells co-transfected with miR-71 or miR-263 overexpression vectors and psi-CHECK2 vectors containing wild-type (WT) or mutant (MT, the 8 nt of the region that corresponds to the miRNA seed mutated) target gene sequences of CHS1 (A) and CHT10 (B) ( n = 6). (C, D) RIP was performed with an anti-Ago-1 antibody. qPCR analysis was performed to amplify the CHS1 and CHT10 mRNAs from the Ago-1 immunoprecipitates from extracts of integument tissue treated with agomir-71 or agomir-263 48 h later compared with the agomir controls (agomir-NC). The data for the luciferase activities and qPCR analyses are presented as means ± SEM ( n = 6). * p

    Techniques Used: Luciferase, Transfection, Over Expression, Mutagenesis, Real-time Polymerase Chain Reaction

    35) Product Images from "The Egalitarian binding partners Dynein light chain and Bicaudal-D act sequentially to link mRNA to the Dynein motor"

    Article Title: The Egalitarian binding partners Dynein light chain and Bicaudal-D act sequentially to link mRNA to the Dynein motor

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.176529

    Artificial dimerization of Egl_2pt restores RNA and BicD binding. (A) Western blot analysis of co-precipitation using S2 cells transfected with: Egl_2pt-GFP and Egl_2pt-FLAG, GFP and Egl_2pt-Zip-FLAG, and Egl_2pt-Zip-GFP and Egl_2pt-Zip-FLAG. Zip refers to a leucine zipper motif. Insertion of the leucine zipper restores Egl_2pt dimerization. (B) Co-precipitation using S2 cells transfected with: GFP, Egl_wt-GFP, Egl_2pt-GFP and Egl_2pt-Zip-GFP. Egl_2pt-Zip-GFP is able to associate with BicD. Asterisk indicates a degradation product that is consistently seen in the Egl_2pt-Zip-GFP lane. (C) Western blot analysis of ILS or ILS-AS binding in S2 cell lysates expressing the indicated constructs. The bound proteins were analyzed by blotting using the FLAG antibody. A total fraction is also shown. (C′) The amount of Egl that co-precipitated with the RNA-bound beads was quantified from four independent biological replicates. Values were normalized to the level of Egl_wt that co-precipitated with ILS and to the expression level of the respective construct in the total lysate. RNA binding activity is restored with Egl_2pt-zip. Data are mean±s.d. *** P
    Figure Legend Snippet: Artificial dimerization of Egl_2pt restores RNA and BicD binding. (A) Western blot analysis of co-precipitation using S2 cells transfected with: Egl_2pt-GFP and Egl_2pt-FLAG, GFP and Egl_2pt-Zip-FLAG, and Egl_2pt-Zip-GFP and Egl_2pt-Zip-FLAG. Zip refers to a leucine zipper motif. Insertion of the leucine zipper restores Egl_2pt dimerization. (B) Co-precipitation using S2 cells transfected with: GFP, Egl_wt-GFP, Egl_2pt-GFP and Egl_2pt-Zip-GFP. Egl_2pt-Zip-GFP is able to associate with BicD. Asterisk indicates a degradation product that is consistently seen in the Egl_2pt-Zip-GFP lane. (C) Western blot analysis of ILS or ILS-AS binding in S2 cell lysates expressing the indicated constructs. The bound proteins were analyzed by blotting using the FLAG antibody. A total fraction is also shown. (C′) The amount of Egl that co-precipitated with the RNA-bound beads was quantified from four independent biological replicates. Values were normalized to the level of Egl_wt that co-precipitated with ILS and to the expression level of the respective construct in the total lysate. RNA binding activity is restored with Egl_2pt-zip. Data are mean±s.d. *** P

    Techniques Used: Binding Assay, Western Blot, Transfection, Expressing, Construct, RNA Binding Assay, Activity Assay

    Dlc is required for Egl dimerization. (A) Western blot analysis of co-immunoprecipitation using ovarian lysates prepared from strains expressing Khc-RFP, Egl_wt-RFP, Egl_2pt-RFP or Egl_4e-RFP. The strains also expressed endogenous Egl. The total fraction is shown. Egl_wt-RFP and Egl_4e-RFP were able to co-precipitate endogenous Egl, whereas Khc-RFP and Egl_2pt-RFP were not. (B) Co-precipitation using S2 cells transfected with: GFP and Egl_wt-FLAG, Egl_wt-GFP and Egl_wt-FLAG, GFP and Egl_2pt-FLAG, and Egl_2pt-GFP and Egl_2pt-FLAG. The total fraction is shown. Egl_2pt is dimerization defective. (C) Co-precipitation using S2 cells transfected with: GFP, Egl_wt-GFP, and Egl_delRBD-GFP (an Egl construct lacking the RNA binding domain). Bound and total fractions are shown. Deletion of the RNA binding domain compromises the Egl-BicD interaction but not the Egl-Dlc interaction. (D) Western blot analysis of ILS or ILS-AS binding in ovarian lysate preparations from strains expressing a control shRNA against the white gene (con shRNA) or an shRNA against dlc . The total fraction is also shown. (D′) The amount of Egl in the respective pellets was quantified from four independent biological replicates. Values were normalized to the level of Egl detected in the ILS fraction from the control sample. Depletion of Dlc compromises the ability of Egl to associate with ILS . (E) Western blot analysis of co-immunoprecipitation using either an antibody against GST or BicD. Bound and total fractions are shown. The amount of Egl that co-precipitated with BicD was quantified from three independent biological replicates. The values were normalized to the level of co-precipitating Egl in the control sample and to the level of BicD that was immunoprecipitated under each condition. Depletion of Dlc reduces the Egl-BicD interaction. Data are mean±s.d. *** P
    Figure Legend Snippet: Dlc is required for Egl dimerization. (A) Western blot analysis of co-immunoprecipitation using ovarian lysates prepared from strains expressing Khc-RFP, Egl_wt-RFP, Egl_2pt-RFP or Egl_4e-RFP. The strains also expressed endogenous Egl. The total fraction is shown. Egl_wt-RFP and Egl_4e-RFP were able to co-precipitate endogenous Egl, whereas Khc-RFP and Egl_2pt-RFP were not. (B) Co-precipitation using S2 cells transfected with: GFP and Egl_wt-FLAG, Egl_wt-GFP and Egl_wt-FLAG, GFP and Egl_2pt-FLAG, and Egl_2pt-GFP and Egl_2pt-FLAG. The total fraction is shown. Egl_2pt is dimerization defective. (C) Co-precipitation using S2 cells transfected with: GFP, Egl_wt-GFP, and Egl_delRBD-GFP (an Egl construct lacking the RNA binding domain). Bound and total fractions are shown. Deletion of the RNA binding domain compromises the Egl-BicD interaction but not the Egl-Dlc interaction. (D) Western blot analysis of ILS or ILS-AS binding in ovarian lysate preparations from strains expressing a control shRNA against the white gene (con shRNA) or an shRNA against dlc . The total fraction is also shown. (D′) The amount of Egl in the respective pellets was quantified from four independent biological replicates. Values were normalized to the level of Egl detected in the ILS fraction from the control sample. Depletion of Dlc compromises the ability of Egl to associate with ILS . (E) Western blot analysis of co-immunoprecipitation using either an antibody against GST or BicD. Bound and total fractions are shown. The amount of Egl that co-precipitated with BicD was quantified from three independent biological replicates. The values were normalized to the level of co-precipitating Egl in the control sample and to the level of BicD that was immunoprecipitated under each condition. Depletion of Dlc reduces the Egl-BicD interaction. Data are mean±s.d. *** P

    Techniques Used: Western Blot, Immunoprecipitation, Expressing, Transfection, Construct, RNA Binding Assay, Binding Assay, shRNA

    36) Product Images from "Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia"

    Article Title: Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-77159-y

    Knockdown of GADD45 exacerbates chromosome segregation defects in Drosophila S2 cells with concomitant Shot knockdown. ( A – D ) Drosophila S2 cells treated without (Control) or with RNAi targeting shot and gadd45 were paraformaldehyde fixed and stained for phosphohistone-H3 (PH3, green) and a-Tubulin (red). Yellow arrows indicate lagging or bridged chromosomes in anaphase (ana) and telophase (telo), respectively. Images are representative of at least 30 cells. ( E ) Graph depicts percentage of cells with normal or Bridged/Lagging chromosomes for indicated treatment conditions. *p
    Figure Legend Snippet: Knockdown of GADD45 exacerbates chromosome segregation defects in Drosophila S2 cells with concomitant Shot knockdown. ( A – D ) Drosophila S2 cells treated without (Control) or with RNAi targeting shot and gadd45 were paraformaldehyde fixed and stained for phosphohistone-H3 (PH3, green) and a-Tubulin (red). Yellow arrows indicate lagging or bridged chromosomes in anaphase (ana) and telophase (telo), respectively. Images are representative of at least 30 cells. ( E ) Graph depicts percentage of cells with normal or Bridged/Lagging chromosomes for indicated treatment conditions. *p

    Techniques Used: Staining

    GADD45 and Ask1 are required for cell cycle arrest in Drosophila S2 cells following Shot knockdown. ( A – C ) Drosophila S2 cells stably expressing GFP:CID and mCherry: α-Tubulin were treated without (Control) or with RNAi targeting shot and gadd45 . Cells were imaged from prior to nuclear envelope breakdown (NEBD) through anaphase onset. Images are representative of at least 10 cells. Timestamps are relative to NEBD. ( D ) Plots show timing from NEBD to anaphase onset for each recorded cell for the indicated treatment condition. Cells not progressing into anaphase after 3 h were considered to have undergone mitotic arrest (“Arrest”; indicated by percentages listed at top). *p
    Figure Legend Snippet: GADD45 and Ask1 are required for cell cycle arrest in Drosophila S2 cells following Shot knockdown. ( A – C ) Drosophila S2 cells stably expressing GFP:CID and mCherry: α-Tubulin were treated without (Control) or with RNAi targeting shot and gadd45 . Cells were imaged from prior to nuclear envelope breakdown (NEBD) through anaphase onset. Images are representative of at least 10 cells. Timestamps are relative to NEBD. ( D ) Plots show timing from NEBD to anaphase onset for each recorded cell for the indicated treatment condition. Cells not progressing into anaphase after 3 h were considered to have undergone mitotic arrest (“Arrest”; indicated by percentages listed at top). *p

    Techniques Used: Stable Transfection, Expressing

    37) Product Images from "Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function"

    Article Title: Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function

    Journal: bioRxiv

    doi: 10.1101/2021.04.29.441910

    dCNBP controls polyamine metabolism through the binding and the translational control of dOdc mRNA. A. d Odc1 mRNA levels (qPCR), normalized with the housekeeping RPL11 mRNA third instar larvae bearing C179GAL4 driver alone or in combination with UAS- 2XdCNBP RNAi . ns: not significative in unpaired t-test. Dots correspond to four independent biological replicates; bars indicate the mean and SEM. B. CNBP binds Odc1 mRNA. qRT-PCR analysis on mRNAs immunoprecipitated by anti-dCNBP antibody or control IgG antisera in S2 cells extracts (left), or in dCNBP-depleted (or not) larval extracts (right). The results are indicated as fold difference, relative to IgG. Error bars represent SEM of three independent biological experiments; * P
    Figure Legend Snippet: dCNBP controls polyamine metabolism through the binding and the translational control of dOdc mRNA. A. d Odc1 mRNA levels (qPCR), normalized with the housekeeping RPL11 mRNA third instar larvae bearing C179GAL4 driver alone or in combination with UAS- 2XdCNBP RNAi . ns: not significative in unpaired t-test. Dots correspond to four independent biological replicates; bars indicate the mean and SEM. B. CNBP binds Odc1 mRNA. qRT-PCR analysis on mRNAs immunoprecipitated by anti-dCNBP antibody or control IgG antisera in S2 cells extracts (left), or in dCNBP-depleted (or not) larval extracts (right). The results are indicated as fold difference, relative to IgG. Error bars represent SEM of three independent biological experiments; * P

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunoprecipitation

    38) Product Images from "A long non-coding RNA in the let-7 complex acting as a potent and specific death effector of cancer cells"

    Article Title: A long non-coding RNA in the let-7 complex acting as a potent and specific death effector of cancer cells

    Journal: bioRxiv

    doi: 10.1101/2021.07.16.452600

    Cell death was induced by RNA molecules. ( A - C ) Light microscope images showing ph 505 culture cells treated with let-A/medium ( A ), mCherry/medium ( B ), or UV medium ( C ). Scale bars are 20 μm. ( D ) Light microscope pictures showing ph 505 culture cells treated with purified RNA fraction from mCherry/medium. ( E ) Light microscope pictures showing ph 505 culture cells treated with purified DNA fraction from mCherry/medium. ( F ) Light microscope pictures showing ph 505 culture cells treated with purified RNA fraction from let-A/medium. ( G ) Light microscope pictures showing ph 505 culture cells treated with purified DNA fraction from let-A/medium. Scale bars are 40 μm. ( H ) Cell viability measurements of ph 505 cells when RNase or DNase were added to the medium during the induction of let-A or mCherry. Note that adding RNase could significantly increase cell viability in the culture, but DNase could not. ( I ) Cell viability measurements of ph 505 cells treated with RNAs purified from let-A, let-B, I1B , or mCherry/medium. Only purified RNA from let-A/medium was toxic to the cells. ( J ) Cell viability measurements of S2 cells treated with RNAs purified from let-A, let-B, I1B , or mCherry/medium. Purified RNA from let-A/medium was not toxic to S2 Cells. n.s. p ≥ 0.05, * p
    Figure Legend Snippet: Cell death was induced by RNA molecules. ( A - C ) Light microscope images showing ph 505 culture cells treated with let-A/medium ( A ), mCherry/medium ( B ), or UV medium ( C ). Scale bars are 20 μm. ( D ) Light microscope pictures showing ph 505 culture cells treated with purified RNA fraction from mCherry/medium. ( E ) Light microscope pictures showing ph 505 culture cells treated with purified DNA fraction from mCherry/medium. ( F ) Light microscope pictures showing ph 505 culture cells treated with purified RNA fraction from let-A/medium. ( G ) Light microscope pictures showing ph 505 culture cells treated with purified DNA fraction from let-A/medium. Scale bars are 40 μm. ( H ) Cell viability measurements of ph 505 cells when RNase or DNase were added to the medium during the induction of let-A or mCherry. Note that adding RNase could significantly increase cell viability in the culture, but DNase could not. ( I ) Cell viability measurements of ph 505 cells treated with RNAs purified from let-A, let-B, I1B , or mCherry/medium. Only purified RNA from let-A/medium was toxic to the cells. ( J ) Cell viability measurements of S2 cells treated with RNAs purified from let-A, let-B, I1B , or mCherry/medium. Purified RNA from let-A/medium was not toxic to S2 Cells. n.s. p ≥ 0.05, * p

    Techniques Used: Light Microscopy, Purification

    Induced expression of  let-A  resulted in rapid cell death in  ph 505  cells. ( A - F ) Light microscope pictures showing  ph 505  cells cultured in different conditions: ( A ) untransfected; ( B ) silver-treated; ( C ) induced  let-A ; ( D ) induced I1B; ( E ) induced  let-B ; ( F ) induced mCherry. All pictures were taken at two hours after induction. Arrow in ( A ) indicates a  ph 505  cell which has an extended shape and attaches to the plate; arrowhead in ( A ) indicates a round shape floating  ph 505  cell. Asterisks in ( C ) indicate dying cells; dashed arrow in ( C ) indicates a  ph 505  cell with abnormal shape. Scale bars is 20 μm. ( G ) alamarBlue quantification of cell viability at three hours after different RNAs were induced in  ph 505  cells. This assay incorporates an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of medium resulting from cell growth (  Kumar et al., 2018 ). ( H ) alamarBlue quantification of cell viability at three days after different RNAs were induced in  ph 505  cells. ( I ) alamarBlue quantification of cell viability at three hours after different RNAs were induced in S2 cells. n.s. p ≥ 0.05, * p
    Figure Legend Snippet: Induced expression of let-A resulted in rapid cell death in ph 505 cells. ( A - F ) Light microscope pictures showing ph 505 cells cultured in different conditions: ( A ) untransfected; ( B ) silver-treated; ( C ) induced let-A ; ( D ) induced I1B; ( E ) induced let-B ; ( F ) induced mCherry. All pictures were taken at two hours after induction. Arrow in ( A ) indicates a ph 505 cell which has an extended shape and attaches to the plate; arrowhead in ( A ) indicates a round shape floating ph 505 cell. Asterisks in ( C ) indicate dying cells; dashed arrow in ( C ) indicates a ph 505 cell with abnormal shape. Scale bars is 20 μm. ( G ) alamarBlue quantification of cell viability at three hours after different RNAs were induced in ph 505 cells. This assay incorporates an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of medium resulting from cell growth ( Kumar et al., 2018 ). ( H ) alamarBlue quantification of cell viability at three days after different RNAs were induced in ph 505 cells. ( I ) alamarBlue quantification of cell viability at three hours after different RNAs were induced in S2 cells. n.s. p ≥ 0.05, * p

    Techniques Used: Expressing, Light Microscopy, Cell Culture

    39) Product Images from "PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock"

    Article Title: PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock

    Journal: bioRxiv

    doi: 10.1101/2021.12.23.474078

    S47A degradation kinetics is excessively temperature-compensated A) Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were collected at the indicated time points after cycloheximide addition at the indicated temperatures. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B-E) Western Blot quantifications. All experiments were performed with three independent replicates. *p
    Figure Legend Snippet: S47A degradation kinetics is excessively temperature-compensated A) Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were collected at the indicated time points after cycloheximide addition at the indicated temperatures. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B-E) Western Blot quantifications. All experiments were performed with three independent replicates. *p

    Techniques Used: Western Blot, Expressing, Staining

    S47A phosphorylation kinetics is slowed down at cold temperature Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were incubated at the indicated temperatures, and collected at the indicated time points after DBT induction. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B, C and D) Western Blot quantifications. PER signal shown at time point 0 is used as a reference to classify PER isoforms as hypo-phosphorylated. Graphs representing the relative amount of hypo-phosphorylated PER at 18, 25 and 29 0 C for the respective genotypes at different time points after DBT induction. S47A shows increased hypo-phosphorylated PER 6h after DBT induction.*p
    Figure Legend Snippet: S47A phosphorylation kinetics is slowed down at cold temperature Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were incubated at the indicated temperatures, and collected at the indicated time points after DBT induction. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B, C and D) Western Blot quantifications. PER signal shown at time point 0 is used as a reference to classify PER isoforms as hypo-phosphorylated. Graphs representing the relative amount of hypo-phosphorylated PER at 18, 25 and 29 0 C for the respective genotypes at different time points after DBT induction. S47A shows increased hypo-phosphorylated PER 6h after DBT induction.*p

    Techniques Used: Western Blot, Expressing, Incubation, Staining

    40) Product Images from "Activity of Fusion Prophenoloxidase-GFP and Its Potential Applications for Innate Immunity Study"

    Article Title: Activity of Fusion Prophenoloxidase-GFP and Its Potential Applications for Innate Immunity Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064106

    Phenoloxidase in S2 cells over-expressing rPPO3-GFP. DIC and fluorescence images were taken in the absence (A–C; −Cu 2+ ) or presence (D–F; +Cu 2+ ) of Cu 2+ during cell transfection. At 48 h, cells with green fluorescence were detected (A, B, D, E), and samples were then stained for PO activity (C, F) as described in Figure 1A–F . When Cu 2+ was absent, some cells with green fluorescence (B) were stained dark brown (C), which is different from rPPO1-GFP ( Figure 1 ) and rPPO2-GFP ( Figure 2 ). There were also cells with green fluorescence that were not stained for PO activity (C). When Cu 2+ was present, cells auto-melanized (see arrow in D). The arrowheads point to cells (E) with green fluorescence that were positively stained. (G-J) rPPO3 and rPPO3-GFP were over-expressed in S2 cells in the absence (G, H) or presence (I, J) of Cu 2+ for 48 h for comparison. In the absence of Cu 2+ , there were still many cells with PO activity (G, H), and many cells were also fluorescent but without PO activity (G). When Cu 2+ was present during transfection, many cells expressing rPPO3 or rPPO3-GFP were stained dark brown (I, J), and some of these cells were auto-melanized as shown in (D). (K) rPO3 activity assay as shown in Figure 1K . When Cu 2+ was present, rPPO3 had almost the same enzyme activity as rPPO3-GFP, and these levels were significantly higher than when Cu 2+ was not added during transfection. Columns represent the mean of individual measurements ± S.E.M (n = 3). Significant differences were calculated using the unpaired t test. (L) Western blot showing that rPPO3-GFP and rPPO3 protein expression occurred regardless of the presence or absence of Cu 2+ . However, due to auto-melanization, in the presence of Cu 2+ some rPPO3 and rPPO3-GFP form large complexes that were not easily separated by SDS-PAGE. Thus, the amounts of rPPO3 and rPPO3-GFP seem lower than those without Cu 2+ added. Bar: 20 µm.
    Figure Legend Snippet: Phenoloxidase in S2 cells over-expressing rPPO3-GFP. DIC and fluorescence images were taken in the absence (A–C; −Cu 2+ ) or presence (D–F; +Cu 2+ ) of Cu 2+ during cell transfection. At 48 h, cells with green fluorescence were detected (A, B, D, E), and samples were then stained for PO activity (C, F) as described in Figure 1A–F . When Cu 2+ was absent, some cells with green fluorescence (B) were stained dark brown (C), which is different from rPPO1-GFP ( Figure 1 ) and rPPO2-GFP ( Figure 2 ). There were also cells with green fluorescence that were not stained for PO activity (C). When Cu 2+ was present, cells auto-melanized (see arrow in D). The arrowheads point to cells (E) with green fluorescence that were positively stained. (G-J) rPPO3 and rPPO3-GFP were over-expressed in S2 cells in the absence (G, H) or presence (I, J) of Cu 2+ for 48 h for comparison. In the absence of Cu 2+ , there were still many cells with PO activity (G, H), and many cells were also fluorescent but without PO activity (G). When Cu 2+ was present during transfection, many cells expressing rPPO3 or rPPO3-GFP were stained dark brown (I, J), and some of these cells were auto-melanized as shown in (D). (K) rPO3 activity assay as shown in Figure 1K . When Cu 2+ was present, rPPO3 had almost the same enzyme activity as rPPO3-GFP, and these levels were significantly higher than when Cu 2+ was not added during transfection. Columns represent the mean of individual measurements ± S.E.M (n = 3). Significant differences were calculated using the unpaired t test. (L) Western blot showing that rPPO3-GFP and rPPO3 protein expression occurred regardless of the presence or absence of Cu 2+ . However, due to auto-melanization, in the presence of Cu 2+ some rPPO3 and rPPO3-GFP form large complexes that were not easily separated by SDS-PAGE. Thus, the amounts of rPPO3 and rPPO3-GFP seem lower than those without Cu 2+ added. Bar: 20 µm.

    Techniques Used: Expressing, Fluorescence, Transfection, Staining, Activity Assay, Western Blot, SDS Page

    Phenoloxidase in S2 cells over-expressing rPPO2-GFP. DIC and fluorescence images were taken in the absence (A–C; −Cu 2+ ) or presence (D–F; +Cu 2+ ) of Cu 2+ during cell transfection. In both cases, cells with green fluorescence were detected (A, B, D, E), and in the presence of Cu 2+ those fluorescent cells displayed PO activity (dark brown) when incubated with dopamine dissolved in 30% ethanol (F). (G–J) rPPO2 and rPPO2-GFP were over-expressed in S2 cells in the absence (G, H) or presence (I, J) of Cu 2+ for 48 h. When Cu 2+ was not added, no cells stained for PO activity (G, H) even though some cells expressed rPPO2-GFP (green fluorescence). When Cu 2+ was added, many cells stained to show PO activity (I, J; brown pigment), and no fluorescent cells were observed after PO staining due to quenching by melanin. (K) rPO2 activity assay as shown in Figure 1K . When Cu 2+ was added, rPPO2 had the same enzyme activity as rPPO2-GFP. No enzyme activity was detected if Cu 2+ was not added. Columns represent the mean of individual measurements ± S.E.M (n = 3). Significant differences were calculated using the unpaired t test. (L) Western blot showing that rPPO2-GFP and rPPO2 protein expression occurred regardless of the presence or absence of Cu 2+ . Bar: 20 µm.
    Figure Legend Snippet: Phenoloxidase in S2 cells over-expressing rPPO2-GFP. DIC and fluorescence images were taken in the absence (A–C; −Cu 2+ ) or presence (D–F; +Cu 2+ ) of Cu 2+ during cell transfection. In both cases, cells with green fluorescence were detected (A, B, D, E), and in the presence of Cu 2+ those fluorescent cells displayed PO activity (dark brown) when incubated with dopamine dissolved in 30% ethanol (F). (G–J) rPPO2 and rPPO2-GFP were over-expressed in S2 cells in the absence (G, H) or presence (I, J) of Cu 2+ for 48 h. When Cu 2+ was not added, no cells stained for PO activity (G, H) even though some cells expressed rPPO2-GFP (green fluorescence). When Cu 2+ was added, many cells stained to show PO activity (I, J; brown pigment), and no fluorescent cells were observed after PO staining due to quenching by melanin. (K) rPO2 activity assay as shown in Figure 1K . When Cu 2+ was added, rPPO2 had the same enzyme activity as rPPO2-GFP. No enzyme activity was detected if Cu 2+ was not added. Columns represent the mean of individual measurements ± S.E.M (n = 3). Significant differences were calculated using the unpaired t test. (L) Western blot showing that rPPO2-GFP and rPPO2 protein expression occurred regardless of the presence or absence of Cu 2+ . Bar: 20 µm.

    Techniques Used: Expressing, Fluorescence, Transfection, Activity Assay, Incubation, Staining, Western Blot

    Co-expression of rPPO2-DsRed with either rPPO1-GFP or rPPO3-GFP. Plasmids containing rPPO1-GFP and rPPO2-DsRed (A-B) or rPPO3-GFP and rPPO2-DsRed (D-E) were co-transfected into S2 cells in the absence of Cu 2+ . Cells with green fluorescence (arrows) expressed rPPO1-GFP or rPPO3-GFP, and cells with red fluorescence (asterisks) expressed rPPO2-DsRed. Cells with yellow fluorescence (arrowheads) co-expressed rPPO2-DsRed and either rPPO1-GFP (B) or rPPO3-GFP (E). All images were taken using green and red filters, and were then merged. (C-F) Among PPO positively stained S2 cells, over 60% had yellow fluorescence, approximately 25% had green fluorescence, and less than 10% had red fluorescence (C, F). Obviously, the ratio of cells with yellow fluorescence is significantly higher than that with red or green fluorescence but not all cells were expressing multiple rPPOs at the same time. Columns represent the mean of individual measurements ± S.E.M (n = 4). Significant differences were calculated using the unpaired t test program. Bar: 20 µm.
    Figure Legend Snippet: Co-expression of rPPO2-DsRed with either rPPO1-GFP or rPPO3-GFP. Plasmids containing rPPO1-GFP and rPPO2-DsRed (A-B) or rPPO3-GFP and rPPO2-DsRed (D-E) were co-transfected into S2 cells in the absence of Cu 2+ . Cells with green fluorescence (arrows) expressed rPPO1-GFP or rPPO3-GFP, and cells with red fluorescence (asterisks) expressed rPPO2-DsRed. Cells with yellow fluorescence (arrowheads) co-expressed rPPO2-DsRed and either rPPO1-GFP (B) or rPPO3-GFP (E). All images were taken using green and red filters, and were then merged. (C-F) Among PPO positively stained S2 cells, over 60% had yellow fluorescence, approximately 25% had green fluorescence, and less than 10% had red fluorescence (C, F). Obviously, the ratio of cells with yellow fluorescence is significantly higher than that with red or green fluorescence but not all cells were expressing multiple rPPOs at the same time. Columns represent the mean of individual measurements ± S.E.M (n = 4). Significant differences were calculated using the unpaired t test program. Bar: 20 µm.

    Techniques Used: Expressing, Transfection, Fluorescence, Staining

    Phenoloxidase in S2 cells over-expressing rPPO1-GFP. DIC and fluorescence images were taken in the absence (A–C; −Cu 2+ ) or presence (D–F; +Cu 2+ ) of Cu 2+ during cell transfection. In both cases, cells with green fluorescence were detected (A, B, D, E), and in the presence of Cu 2+ those fluorescent cells displayed PO activity (dark brown) when incubated with dopamine dissolved in 30% ethanol (F). (G-J). Comparison of cells with rPPO1 and rPPO1-GFP expressed. rPPO1 and rPPO1-GFP were over-expressed in S2 cells in the absence (G, H) or presence (I, J) of Cu 2+ during cell transfection. The cells were then stained for PO activity. Images represent DIC and fluorescence overlays. When Cu 2+ was not added, no cells stained for PO activity (G, H) even though some cells expressed rPPO1-GFP (green fluorescence). When Cu 2+ was added, many cells stained for PO activity (I, J; brown pigment), and no fluorescent cells were observed after PO staining due to quenching by melanin. (K) Comparison of rPPO1 and rPPO1-GFP enzyme activities. The amounts of rPPO1 and PPO1-GFP in S2 cell lysates were normalized and determined using purified rPPO1 as a standard by Western blot. Ethanol was used for enzyme activation. When Cu 2+ was added, rPPO1 had significantly higher enzyme activity than rPPO1-GFP. No enzyme activities were detected if Cu 2+ was not added, which is in agreement with the cell staining shown in (G-J). Columns represent the mean of individual measurements ± S.E.M (n = 3). Significant differences were calculated using the unpaired t test. (L) A Western blot showed that rPPO1-GFP and rPPO1 protein expression occurred regardless of the presence or absence of Cu 2+ . Bar: 20 µm.
    Figure Legend Snippet: Phenoloxidase in S2 cells over-expressing rPPO1-GFP. DIC and fluorescence images were taken in the absence (A–C; −Cu 2+ ) or presence (D–F; +Cu 2+ ) of Cu 2+ during cell transfection. In both cases, cells with green fluorescence were detected (A, B, D, E), and in the presence of Cu 2+ those fluorescent cells displayed PO activity (dark brown) when incubated with dopamine dissolved in 30% ethanol (F). (G-J). Comparison of cells with rPPO1 and rPPO1-GFP expressed. rPPO1 and rPPO1-GFP were over-expressed in S2 cells in the absence (G, H) or presence (I, J) of Cu 2+ during cell transfection. The cells were then stained for PO activity. Images represent DIC and fluorescence overlays. When Cu 2+ was not added, no cells stained for PO activity (G, H) even though some cells expressed rPPO1-GFP (green fluorescence). When Cu 2+ was added, many cells stained for PO activity (I, J; brown pigment), and no fluorescent cells were observed after PO staining due to quenching by melanin. (K) Comparison of rPPO1 and rPPO1-GFP enzyme activities. The amounts of rPPO1 and PPO1-GFP in S2 cell lysates were normalized and determined using purified rPPO1 as a standard by Western blot. Ethanol was used for enzyme activation. When Cu 2+ was added, rPPO1 had significantly higher enzyme activity than rPPO1-GFP. No enzyme activities were detected if Cu 2+ was not added, which is in agreement with the cell staining shown in (G-J). Columns represent the mean of individual measurements ± S.E.M (n = 3). Significant differences were calculated using the unpaired t test. (L) A Western blot showed that rPPO1-GFP and rPPO1 protein expression occurred regardless of the presence or absence of Cu 2+ . Bar: 20 µm.

    Techniques Used: Expressing, Fluorescence, Transfection, Activity Assay, Incubation, Staining, Purification, Western Blot, Activation Assay

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    Thermo Fisher blasticidin s hcl
    Exclusion of potential interference between different resistance genes using strains showing either Zeocin, nourseothricin or <t>blasticidin-S</t> resistance. (A) Nine blasticidin-S-resistant P. tricornutum colonies were spread on plates containing Zeocin or nourseothricin and on a control plate containing blasticidin-S. (B) Nine Zeocin-resistant colonies were spread on blasticidin-S plates and on control plates with Zeocin. (C) Nine nourseothricin-resistant colonies were spread on plates with blasticidin-S and on control plates with nourseothricin. The cells were able to survive only on the appropriate antibiotics. (+) = growth of the cells; (−) = no growth.
    Blasticidin S Hcl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dcv infected drosophila s2 cells
    The heat shock response is dynamic and requires viral replication in Drosophila S2 cells. Expression of the genes encoding the heat shock proteins Hsp70, Hsp23, Hsp26, and the Heat shock transcription factor (Hsf) was monitored at the indicated time points by RT-qPCR after infection with ( a ) <t>DCV,</t> ( b ) CrPV or ( c ) IIV-6 (MOI = 10). ( d ) <t>S2</t> cells were inoculated with UV-inactivated viruses and gene expression was measured at 24, 16 or 48 hpi with DCV, CrPV and IIV-6, respectively. Expression of the gene of interest was normalized to the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection. Data are mean and s.d. of three independent infections. Student’s t-tests were used to compare virus-infected samples to mock infections (* P
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    Thermo Fisher mouse mc1r
    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without <t>MC1R</t> RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p
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    Exclusion of potential interference between different resistance genes using strains showing either Zeocin, nourseothricin or blasticidin-S resistance. (A) Nine blasticidin-S-resistant P. tricornutum colonies were spread on plates containing Zeocin or nourseothricin and on a control plate containing blasticidin-S. (B) Nine Zeocin-resistant colonies were spread on blasticidin-S plates and on control plates with Zeocin. (C) Nine nourseothricin-resistant colonies were spread on plates with blasticidin-S and on control plates with nourseothricin. The cells were able to survive only on the appropriate antibiotics. (+) = growth of the cells; (−) = no growth.

    Journal: PeerJ

    Article Title: Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum

    doi: 10.7717/peerj.5884

    Figure Lengend Snippet: Exclusion of potential interference between different resistance genes using strains showing either Zeocin, nourseothricin or blasticidin-S resistance. (A) Nine blasticidin-S-resistant P. tricornutum colonies were spread on plates containing Zeocin or nourseothricin and on a control plate containing blasticidin-S. (B) Nine Zeocin-resistant colonies were spread on blasticidin-S plates and on control plates with Zeocin. (C) Nine nourseothricin-resistant colonies were spread on plates with blasticidin-S and on control plates with nourseothricin. The cells were able to survive only on the appropriate antibiotics. (+) = growth of the cells; (−) = no growth.

    Article Snippet: Blasticidin-S (R21001; Thermo Fisher, Waltham, MA, USA) and tunicamycin (Abcam, Cambridge, UK) were prepared as stock solutions of 10 mg/ml in water.

    Techniques:

    Incubation of P. tricornutum on plates containing different concentrations of blasticidin-S. 2.5 × 10 7 P. tricornutum cells were spread on plates (25% salinity of seawater) and containing different concentrations of blasticidin-S. At concentrations between 0 and two μg/ml of blasticidin-S (A–E) we observed a lawn of cells. At 2.5 μg/ml (F) and 3.0 μg/ml (G) we observed scattered growth, and above 3.5 μg/ml (H) no growth was observed. Magnified areas in the picture are indicated by an arrow.

    Journal: PeerJ

    Article Title: Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum

    doi: 10.7717/peerj.5884

    Figure Lengend Snippet: Incubation of P. tricornutum on plates containing different concentrations of blasticidin-S. 2.5 × 10 7 P. tricornutum cells were spread on plates (25% salinity of seawater) and containing different concentrations of blasticidin-S. At concentrations between 0 and two μg/ml of blasticidin-S (A–E) we observed a lawn of cells. At 2.5 μg/ml (F) and 3.0 μg/ml (G) we observed scattered growth, and above 3.5 μg/ml (H) no growth was observed. Magnified areas in the picture are indicated by an arrow.

    Article Snippet: Blasticidin-S (R21001; Thermo Fisher, Waltham, MA, USA) and tunicamycin (Abcam, Cambridge, UK) were prepared as stock solutions of 10 mg/ml in water.

    Techniques: Incubation

    Plasmid map of the vector pPTbsr. ) but includes the resistance gene blasticidin-S deaminase ( bsr ) instead of the Zeocin resistance cassette Sh Ble. MCS, multiple cloning site; fcpA/B, fucoxanthin-chlorophyll-binding protein A/B; prom, promoter; term, terminator.

    Journal: PeerJ

    Article Title: Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum

    doi: 10.7717/peerj.5884

    Figure Lengend Snippet: Plasmid map of the vector pPTbsr. ) but includes the resistance gene blasticidin-S deaminase ( bsr ) instead of the Zeocin resistance cassette Sh Ble. MCS, multiple cloning site; fcpA/B, fucoxanthin-chlorophyll-binding protein A/B; prom, promoter; term, terminator.

    Article Snippet: Blasticidin-S (R21001; Thermo Fisher, Waltham, MA, USA) and tunicamycin (Abcam, Cambridge, UK) were prepared as stock solutions of 10 mg/ml in water.

    Techniques: Plasmid Preparation, Clone Assay, Binding Assay

    The heat shock response is dynamic and requires viral replication in Drosophila S2 cells. Expression of the genes encoding the heat shock proteins Hsp70, Hsp23, Hsp26, and the Heat shock transcription factor (Hsf) was monitored at the indicated time points by RT-qPCR after infection with ( a ) DCV, ( b ) CrPV or ( c ) IIV-6 (MOI = 10). ( d ) S2 cells were inoculated with UV-inactivated viruses and gene expression was measured at 24, 16 or 48 hpi with DCV, CrPV and IIV-6, respectively. Expression of the gene of interest was normalized to the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection. Data are mean and s.d. of three independent infections. Student’s t-tests were used to compare virus-infected samples to mock infections (* P

    Journal: Scientific Reports

    Article Title: The heat shock response restricts virus infection in Drosophila

    doi: 10.1038/srep12758

    Figure Lengend Snippet: The heat shock response is dynamic and requires viral replication in Drosophila S2 cells. Expression of the genes encoding the heat shock proteins Hsp70, Hsp23, Hsp26, and the Heat shock transcription factor (Hsf) was monitored at the indicated time points by RT-qPCR after infection with ( a ) DCV, ( b ) CrPV or ( c ) IIV-6 (MOI = 10). ( d ) S2 cells were inoculated with UV-inactivated viruses and gene expression was measured at 24, 16 or 48 hpi with DCV, CrPV and IIV-6, respectively. Expression of the gene of interest was normalized to the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection. Data are mean and s.d. of three independent infections. Student’s t-tests were used to compare virus-infected samples to mock infections (* P

    Article Snippet: The heat shock response is induced in DCV-infected Drosophila S2 cells To identify novel factors or processes involved in antiviral defence in Drosophila , we generated transcriptional profiles of DCV-infected Drosophila S2 cells at 8 and 24 hours post-infection (hpi) using Affymetrix GeneChip microarrays ( ).

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Microarray analysis of DCV-infected Drosophila S2 cells. ( a ) Overview of the experimental workflow. S2 cells were infected with DCV (MOI = 10) or mock-infected with Schneider’s medium, and RNA was extracted at 8 and 24 hours post-infection (hpi) for microarray analyses. Figure drawn by S.H. Merkling. ( b ) Number of differentially expressed genes at 8 and 24 hpi (fold change ≥2 relative to mock infection). ( c ) Venn diagram representing the overlap between differentially induced genes after DCV infection at 8 and 24 hpi. ( d , e ) Gene ontology (GO) analysis of the genes that are upregulated ≥2-fold at ( d ) 8 hpi and ( e ) 24 hpi. All significantly enriched level 4 GO terms are shown ( P

    Journal: Scientific Reports

    Article Title: The heat shock response restricts virus infection in Drosophila

    doi: 10.1038/srep12758

    Figure Lengend Snippet: Microarray analysis of DCV-infected Drosophila S2 cells. ( a ) Overview of the experimental workflow. S2 cells were infected with DCV (MOI = 10) or mock-infected with Schneider’s medium, and RNA was extracted at 8 and 24 hours post-infection (hpi) for microarray analyses. Figure drawn by S.H. Merkling. ( b ) Number of differentially expressed genes at 8 and 24 hpi (fold change ≥2 relative to mock infection). ( c ) Venn diagram representing the overlap between differentially induced genes after DCV infection at 8 and 24 hpi. ( d , e ) Gene ontology (GO) analysis of the genes that are upregulated ≥2-fold at ( d ) 8 hpi and ( e ) 24 hpi. All significantly enriched level 4 GO terms are shown ( P

    Article Snippet: The heat shock response is induced in DCV-infected Drosophila S2 cells To identify novel factors or processes involved in antiviral defence in Drosophila , we generated transcriptional profiles of DCV-infected Drosophila S2 cells at 8 and 24 hours post-infection (hpi) using Affymetrix GeneChip microarrays ( ).

    Techniques: Microarray, Infection

    Binding of Upd to Dally. ( A ) Upd-HA was expressed in S2 cells with or without a secreted form of Dally-Myc. Fractions from the cell pellet (c) and supernatant (s) were probed with anti-HA antibody. ( B ) Upd-HA was expressed in S2 cells with or without

    Journal: Development (Cambridge, England)

    Article Title: Glypicans regulate JAK/STAT signaling and distribution of the Unpaired morphogen

    doi: 10.1242/dev.078055

    Figure Lengend Snippet: Binding of Upd to Dally. ( A ) Upd-HA was expressed in S2 cells with or without a secreted form of Dally-Myc. Fractions from the cell pellet (c) and supernatant (s) were probed with anti-HA antibody. ( B ) Upd-HA was expressed in S2 cells with or without

    Article Snippet: Drosophila S2 cells were grown in Schneider's medium (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Binding Assay

    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Journal: PLoS ONE

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    doi: 10.1371/journal.pone.0056004

    Figure Lengend Snippet: (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Article Snippet: MC1R RNA Interference (RNAi) The chemically synthesized MC1R siRNA (small interfering RNA) duplexes ( ) against mouse MC1R (s1, s2 and s3) were purchased from Ambion (GenePharm Co. Ltd. Shanghai, China).

    Techniques: Activity Assay, Cell Culture, Transfection, MTT Assay

    (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Journal: PLoS ONE

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    doi: 10.1371/journal.pone.0056004

    Figure Lengend Snippet: (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Article Snippet: MC1R RNA Interference (RNAi) The chemically synthesized MC1R siRNA (small interfering RNA) duplexes ( ) against mouse MC1R (s1, s2 and s3) were purchased from Ambion (GenePharm Co. Ltd. Shanghai, China).

    Techniques: Sequencing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control