drosophila s2 cells  (Thermo Fisher)


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    Thermo Fisher drosophila s2 cells
    Binding of Upd to Dally. ( A ) Upd-HA was expressed in <t>S2</t> 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
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    Images

    1) Product Images from "Glypicans regulate JAK/STAT signaling and distribution of the Unpaired morphogen"

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

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.078055

    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
    Figure Legend 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

    Techniques Used: Binding Assay

    2) Product Images from "Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling"

    Article Title: Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.09.011

    Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .
    Figure Legend Snippet: Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .

    Techniques Used: Inhibition, Radial Immuno Diffusion, Activation Assay, Recombinant, Quantitative RT-PCR, Expressing, Injection, Real-time Polymerase Chain Reaction

    PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p
    Figure Legend Snippet: PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p

    Techniques Used: Radial Immuno Diffusion, Expressing, Mutagenesis, Positive Control, Transfection, Fluorescence

    3) Product Images from "A Map of Drosophila melanogaster Small Nuclear RNA-activating Protein Complex (DmSNAPc) Domains Involved in Subunit Assembly and DNA Binding *"

    Article Title: A Map of Drosophila melanogaster Small Nuclear RNA-activating Protein Complex (DmSNAPc) Domains Involved in Subunit Assembly and DNA Binding *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.027961

    Domains of DmSNAP50 involved in assembly in vivo with DmSNAP190 and DmSNAP43. A , schematic representation of full-length and truncated DmSNAP50 constructs co-transfected into S2 cells together with constructs expressing full-length untagged DmSNAP190 and DmSNAP43. The shaded area represents the region most conserved between humans and fruit flies. The black rectangles represent FLAG tags at the C or N termini of DmSNAP50. The names of the constructs in the column at the left indicate the extent of the wild type amino acid residues present in the expressed constructs. B , co-overexpression of tagged DmSNAP50 with DmSNAP190 and DmSNAP43 in stably transfected S2 cells. Whole cell extracts from stably transfected cells co-overexpressing all three DmSNAP subunits were run on denaturing gels and DmSNAP subunits were detected by immunoblot analysis. The amount of extract loaded in each lane was normalized so that the intensity of the signal obtained from the tagged DmSNAP50 construct was similar in each lane. The middle panels show full-length or truncated tagged DmSNAP50 detected using monoclonal antibodies against the FLAG epitope. The top and bottom panels ). C , co-purification of DmSNAP190 and DmSNAP43 with full-length and truncated DmSNAP50 constructs following FLAG affinity purification under low-salt conditions. Complexes containing full-length or truncated tagged DmSNAP50 were purified using FLAG antibody beads, and the presence of the individual subunits in the elution fractions was evaluated by immunoblotting. The volume of elution fraction loaded in each lane was normalized so that the intensity of the signal for the DmSNAP50 construct was similar in each lane when detected with FLAG antibodies. DmSNAP190 and DmSNAP43 subunits that co-purified with the FLAG-tagged DmSNAP50 constructs were detected with antibodies prepared against C-terminal peptides of the respective proteins. D , same as C except FLAG affinity purification was carried out under high-salt conditions.
    Figure Legend Snippet: Domains of DmSNAP50 involved in assembly in vivo with DmSNAP190 and DmSNAP43. A , schematic representation of full-length and truncated DmSNAP50 constructs co-transfected into S2 cells together with constructs expressing full-length untagged DmSNAP190 and DmSNAP43. The shaded area represents the region most conserved between humans and fruit flies. The black rectangles represent FLAG tags at the C or N termini of DmSNAP50. The names of the constructs in the column at the left indicate the extent of the wild type amino acid residues present in the expressed constructs. B , co-overexpression of tagged DmSNAP50 with DmSNAP190 and DmSNAP43 in stably transfected S2 cells. Whole cell extracts from stably transfected cells co-overexpressing all three DmSNAP subunits were run on denaturing gels and DmSNAP subunits were detected by immunoblot analysis. The amount of extract loaded in each lane was normalized so that the intensity of the signal obtained from the tagged DmSNAP50 construct was similar in each lane. The middle panels show full-length or truncated tagged DmSNAP50 detected using monoclonal antibodies against the FLAG epitope. The top and bottom panels ). C , co-purification of DmSNAP190 and DmSNAP43 with full-length and truncated DmSNAP50 constructs following FLAG affinity purification under low-salt conditions. Complexes containing full-length or truncated tagged DmSNAP50 were purified using FLAG antibody beads, and the presence of the individual subunits in the elution fractions was evaluated by immunoblotting. The volume of elution fraction loaded in each lane was normalized so that the intensity of the signal for the DmSNAP50 construct was similar in each lane when detected with FLAG antibodies. DmSNAP190 and DmSNAP43 subunits that co-purified with the FLAG-tagged DmSNAP50 constructs were detected with antibodies prepared against C-terminal peptides of the respective proteins. D , same as C except FLAG affinity purification was carried out under high-salt conditions.

    Techniques Used: In Vivo, Construct, Transfection, Expressing, Over Expression, Stable Transfection, FLAG-tag, Copurification, Affinity Purification, Purification

    Domains of DmSNAP43 involved in assembly in vivo with DmSNAP190 and DmSNAP50. A , schematic representation of full-length and truncated DmSNAP43 constructs co-transfected into S2 cells together with constructs expressing full-length untagged DmSNAP190 and DmSNAP50. The shaded area represents the region most conserved between humans and fruit flies. The black rectangles represent FLAG tags at the C or N termini of DmSNAP43. The names of the constructs in the column at the left indicate the extent of the wild type amino acid residues present in the expressed constructs. B , co-overexpression of tagged DmSNAP43 with DmSNAP190 and DmSNAP50 in stably transfected S2 cells. Whole cell extracts from stably transfected cells co-overexpressing all three DmSNAP subunits were run on denaturing gels and DmSNAP subunits were detected by immunoblot analysis. The amount of extract loaded in each lane was normalized so that the intensity of the signal obtained from the tagged DmSNAP43 construct was similar in each lane. The bottom panels show full-length or truncated tagged DmSNAP43 detected using monoclonal antibodies against the FLAG epitope. The top and middle panels ). C , co-purification of DmSNAP190 and DmSNAP50 with full-length and truncated DmSNAP43 constructs following FLAG affinity purification under low-salt conditions. Complexes containing full-length or truncated tagged DmSNAP43 were purified using FLAG antibody beads, and the presence of the individual subunits in the elution fractions was evaluated by immunoblotting. The volume of elution fraction loaded in each lane was normalized so that the intensity of the signal for the DmSNAP43 construct was similar in each lane when detected with FLAG antibodies. DmSNAP190 and DmSNAP50 subunits that co-purified with the FLAG-tagged DmSNAP43 constructs were detected with antibodies prepared against C-terminal peptides of the respective proteins. D , same as C except FLAG affinity purification was carried out under high-salt conditions.
    Figure Legend Snippet: Domains of DmSNAP43 involved in assembly in vivo with DmSNAP190 and DmSNAP50. A , schematic representation of full-length and truncated DmSNAP43 constructs co-transfected into S2 cells together with constructs expressing full-length untagged DmSNAP190 and DmSNAP50. The shaded area represents the region most conserved between humans and fruit flies. The black rectangles represent FLAG tags at the C or N termini of DmSNAP43. The names of the constructs in the column at the left indicate the extent of the wild type amino acid residues present in the expressed constructs. B , co-overexpression of tagged DmSNAP43 with DmSNAP190 and DmSNAP50 in stably transfected S2 cells. Whole cell extracts from stably transfected cells co-overexpressing all three DmSNAP subunits were run on denaturing gels and DmSNAP subunits were detected by immunoblot analysis. The amount of extract loaded in each lane was normalized so that the intensity of the signal obtained from the tagged DmSNAP43 construct was similar in each lane. The bottom panels show full-length or truncated tagged DmSNAP43 detected using monoclonal antibodies against the FLAG epitope. The top and middle panels ). C , co-purification of DmSNAP190 and DmSNAP50 with full-length and truncated DmSNAP43 constructs following FLAG affinity purification under low-salt conditions. Complexes containing full-length or truncated tagged DmSNAP43 were purified using FLAG antibody beads, and the presence of the individual subunits in the elution fractions was evaluated by immunoblotting. The volume of elution fraction loaded in each lane was normalized so that the intensity of the signal for the DmSNAP43 construct was similar in each lane when detected with FLAG antibodies. DmSNAP190 and DmSNAP50 subunits that co-purified with the FLAG-tagged DmSNAP43 constructs were detected with antibodies prepared against C-terminal peptides of the respective proteins. D , same as C except FLAG affinity purification was carried out under high-salt conditions.

    Techniques Used: In Vivo, Construct, Transfection, Expressing, Over Expression, Stable Transfection, FLAG-tag, Copurification, Affinity Purification, Purification

    4) Product Images from "Characterization of a Novel Manduca sexta beta-1, 3-glucan recognition protein (βGRP3) with Multiple Functions"

    Article Title: Characterization of a Novel Manduca sexta beta-1, 3-glucan recognition protein (βGRP3) with Multiple Functions

    Journal: Insect biochemistry and molecular biology

    doi: 10.1016/j.ibmb.2014.06.003

    Analysis of recombinant GFP and βGRP3 purified from Drosophila S2 cells V5-tagged recombinant GFP and βGRP3 proteins were purified from Drosophila S2 cells and analyzed by SDS-PAGE (1.5 μg each protein) (A) and Western blot (0.2 μg each protein) using monoclonal anti-V5 (B) or polyclonal anti-βGRP3 (C) antibody. Lane 1, GFP; lane 2, βGRP3.
    Figure Legend Snippet: Analysis of recombinant GFP and βGRP3 purified from Drosophila S2 cells V5-tagged recombinant GFP and βGRP3 proteins were purified from Drosophila S2 cells and analyzed by SDS-PAGE (1.5 μg each protein) (A) and Western blot (0.2 μg each protein) using monoclonal anti-V5 (B) or polyclonal anti-βGRP3 (C) antibody. Lane 1, GFP; lane 2, βGRP3.

    Techniques Used: Recombinant, Purification, SDS Page, Western Blot

    Binding of βGRP3 to microbial cell wall components Increasing concentrations of recombinant βGRP3 or GFP purified from Drosophila S2 cells were added to 96-well plates coated with LPS-K12 (A), PG-K12 (B), LTA-SA (C), PG-SA (D), LTA-BS (E), PG-BS (F), mannan (G) or laminarin (H), and binding of recombinant proteins to microbial components was determined by plate ELISA using anti-V5 antibody. Recombinant βGRP3 (2 μg/ml) was pre-incubated with increasing amounts of free microbial components and then added to the laminarin-coated 96-well plates (I), and binding of βGRP3 to laminarin was determined by plate ELISA assay. The figures showed total binding of recombinant proteins to microbial components. Each point represents the mean of three individual measurements ± SEM, and the lines in (A–H) represent nonlinear regression calculation of one-site binding curves for βGRP3 (solid line) and GFP (dotted line).
    Figure Legend Snippet: Binding of βGRP3 to microbial cell wall components Increasing concentrations of recombinant βGRP3 or GFP purified from Drosophila S2 cells were added to 96-well plates coated with LPS-K12 (A), PG-K12 (B), LTA-SA (C), PG-SA (D), LTA-BS (E), PG-BS (F), mannan (G) or laminarin (H), and binding of recombinant proteins to microbial components was determined by plate ELISA using anti-V5 antibody. Recombinant βGRP3 (2 μg/ml) was pre-incubated with increasing amounts of free microbial components and then added to the laminarin-coated 96-well plates (I), and binding of βGRP3 to laminarin was determined by plate ELISA assay. The figures showed total binding of recombinant proteins to microbial components. Each point represents the mean of three individual measurements ± SEM, and the lines in (A–H) represent nonlinear regression calculation of one-site binding curves for βGRP3 (solid line) and GFP (dotted line).

    Techniques Used: Binding Assay, Recombinant, Purification, Enzyme-linked Immunosorbent Assay, Incubation

    5) Product Images from "Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts"

    Article Title: Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.113860

    Full length Sns interacts with the SH2 and SH3 domains of Dock, but Sns intra  only interacts with the SH3 domain of Dock.  ( A , B ) Immunoblots of lysates and immunoprecipitates of transfected  Drosophila  S2 cells. (A) Sns full-length interacts Dock full-length
    Figure Legend Snippet: Full length Sns interacts with the SH2 and SH3 domains of Dock, but Sns intra only interacts with the SH3 domain of Dock. ( A , B ) Immunoblots of lysates and immunoprecipitates of transfected Drosophila S2 cells. (A) Sns full-length interacts Dock full-length

    Techniques Used: Western Blot, Transfection

    6) Product Images from "The small G protein Arl8 contributes to lysosomal function and long-range axonal transport in Drosophila"

    Article Title: The small G protein Arl8 contributes to lysosomal function and long-range axonal transport in Drosophila

    Journal: Biology Open

    doi: 10.1242/bio.035964

    Arl8 recruits the Drosophila ortholog of RILP to lysosomes. (A) Immunoblots of lysates from cells treated with control dsRNA, Arl8-1 (dsRNA1) and Arl8-2 (dsRNA2). The blots were probed for Arl8 and for β-actin as an internal control. (B) Confocal micrographs of Drosophila S2 cells expressing GFP-CG1148 or GFP-CG6613 and treated with control dsRNA or two different dsRNAs against Arl8 (dsRNA1 and dsRNA2) as indicated. GFP-CG11448 and GFP-CG6613 were punctate in the majority of cells imaged (80% of 21 and 56% of 16 respectively) with higher expression levels giving a diffuse distribution. After RNAi the punctate distribution for CG11448 was reduced to 12% of 25 (dsRNA1) and 23% of 39 (dsRNA2), whereas CG6613 was not substantially affected with punctate distribution, seen in 40% of 15 (dsRNA1) and 63% of 11 (dsRNA2). Scale bars: 2 μm. (C) Yeast-two-hybrid interactions between the indicated forms of Arl8 and the proteins CG11448 and CG6613.
    Figure Legend Snippet: Arl8 recruits the Drosophila ortholog of RILP to lysosomes. (A) Immunoblots of lysates from cells treated with control dsRNA, Arl8-1 (dsRNA1) and Arl8-2 (dsRNA2). The blots were probed for Arl8 and for β-actin as an internal control. (B) Confocal micrographs of Drosophila S2 cells expressing GFP-CG1148 or GFP-CG6613 and treated with control dsRNA or two different dsRNAs against Arl8 (dsRNA1 and dsRNA2) as indicated. GFP-CG11448 and GFP-CG6613 were punctate in the majority of cells imaged (80% of 21 and 56% of 16 respectively) with higher expression levels giving a diffuse distribution. After RNAi the punctate distribution for CG11448 was reduced to 12% of 25 (dsRNA1) and 23% of 39 (dsRNA2), whereas CG6613 was not substantially affected with punctate distribution, seen in 40% of 15 (dsRNA1) and 63% of 11 (dsRNA2). Scale bars: 2 μm. (C) Yeast-two-hybrid interactions between the indicated forms of Arl8 and the proteins CG11448 and CG6613.

    Techniques Used: Western Blot, Expressing

    Identification of Arl8 interacting proteins by affinity chromatography. (A) Comparison of the spectral counts of proteins isolated by affinity chromatography using GST-Arl8 with mutations that lock the protein in either a GDP or GTP conformations. S2 cell lysates were prepared without detergent. Abundant GTP-specific interactors are labelled: yellow triangles indicating subunits of the NRZ complex, green triangles indicating two subunits of the HOPS complex [Carnation (Car) being Vps33]. Further HOPS subunits [Deep orange (Vps18), Vps11 and Vps39] were present but with fewer spectra (two with GTP, zero with GTP). CG11448 (blue triangle) is the Drosophila . (C) Confocal micrographs of Drosophila S2 cells co-expressing Arl8-RFP and GFP-CG11448. In the lower row the cells were treated with 10 μM nocodazole for 2 h prior to fixation to depolymerise microtubules. (D) Confocal micrograph of S2 cells co-expressing Arl8-RFP (arrows) and Zw10-GFP. (E) Confocal micrographs of S2 cells expressing GFP-CG1148 and stained with antibodies against Arl8 or Rab7. (F) Confocal micrograph of S2 cells expressing GFP-CG6613 and stained with antibodies against Rab7. For panels B-E, at least six images were obtained for each condition with representative examples shown. Scale bars: 2 μm.
    Figure Legend Snippet: Identification of Arl8 interacting proteins by affinity chromatography. (A) Comparison of the spectral counts of proteins isolated by affinity chromatography using GST-Arl8 with mutations that lock the protein in either a GDP or GTP conformations. S2 cell lysates were prepared without detergent. Abundant GTP-specific interactors are labelled: yellow triangles indicating subunits of the NRZ complex, green triangles indicating two subunits of the HOPS complex [Carnation (Car) being Vps33]. Further HOPS subunits [Deep orange (Vps18), Vps11 and Vps39] were present but with fewer spectra (two with GTP, zero with GTP). CG11448 (blue triangle) is the Drosophila . (C) Confocal micrographs of Drosophila S2 cells co-expressing Arl8-RFP and GFP-CG11448. In the lower row the cells were treated with 10 μM nocodazole for 2 h prior to fixation to depolymerise microtubules. (D) Confocal micrograph of S2 cells co-expressing Arl8-RFP (arrows) and Zw10-GFP. (E) Confocal micrographs of S2 cells expressing GFP-CG1148 and stained with antibodies against Arl8 or Rab7. (F) Confocal micrograph of S2 cells expressing GFP-CG6613 and stained with antibodies against Rab7. For panels B-E, at least six images were obtained for each condition with representative examples shown. Scale bars: 2 μm.

    Techniques Used: Affinity Chromatography, Isolation, Expressing, Staining

    7) Product Images from "A large family of Dscam genes with tandemly arrayed 5′ cassettes in Chelicerata"

    Article Title: A large family of Dscam genes with tandemly arrayed 5′ cassettes in Chelicerata

    Journal: Nature Communications

    doi: 10.1038/ncomms11252

    Each variable cassette preceded by a promoter in  sDscam . ( a ) A schematic diagram of the expression of variable cassettes in  M. martensii sDscamβ6 . Symbols used are the same as in  Fig. 1 . Potential promoter elements (PPE) are shown as green circles. ( b ) Analysis of  sDscam  variable cassette promoter in the reporter assays. A portion of the sequence immediately preceding a given variable cassette was cloned into a luciferase reporter construct and subsequently transfected into  Drosophila  S2 cells. The luciferase vector containing the  Drosophila Dscam2  promoter or intronic sequence of  sDscamβ6  served as positive and negative controls, respectively. Schematic diagrams of mutants with the indicated sizes are depicted on the left. The deleted PPEs are shown as dashed circles. Data are expressed as a percentage of the mean±s.d. from three independent experiments. * P
    Figure Legend Snippet: Each variable cassette preceded by a promoter in sDscam . ( a ) A schematic diagram of the expression of variable cassettes in M. martensii sDscamβ6 . Symbols used are the same as in Fig. 1 . Potential promoter elements (PPE) are shown as green circles. ( b ) Analysis of sDscam variable cassette promoter in the reporter assays. A portion of the sequence immediately preceding a given variable cassette was cloned into a luciferase reporter construct and subsequently transfected into Drosophila S2 cells. The luciferase vector containing the Drosophila Dscam2 promoter or intronic sequence of sDscamβ6 served as positive and negative controls, respectively. Schematic diagrams of mutants with the indicated sizes are depicted on the left. The deleted PPEs are shown as dashed circles. Data are expressed as a percentage of the mean±s.d. from three independent experiments. * P

    Techniques Used: Expressing, Sequencing, Clone Assay, Luciferase, Construct, Transfection, Plasmid Preparation

    8) Product Images from "Dop1 enhances conspecific olfactory attraction by inhibiting miR-9a maturation in locusts"

    Article Title: Dop1 enhances conspecific olfactory attraction by inhibiting miR-9a maturation in locusts

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03437-z

    Dop1 does not affect the expression of primary miR-9a (pri-miR-9a) and miR-9a precursor (pre-miR-9a). a Predicted stem–loop structure of pre-miR-9a. b Expression of miR-9a in S2 cells transfected with expression vectors including partial sequence of pri-miR-9a centered pre-miR-9a ( n = 6). c , d Expression levels of pri-miR-9a ( c , n = 8) and pre-miR-9a ( d , n = 8) in total cells after activation and inhibition of Dop1, respectively. e Expression levels of pri-miR-9a in the nucleus after activation and inhibition of Dop1, respectively. U6 was used as a nuclear marker and 18 S rRNA as a cytoplasmic marker. f , g Expression levels of pre-miR-9a in the nucleus ( f , n = 8) and cytoplasm ( g , n = 8) after activation and inhibition of Dop1, respectively. The asterisks outside the strip indicate the significant difference between controls and the treatments through Student’s t -test and presented as the mean ± SEM. ** P
    Figure Legend Snippet: Dop1 does not affect the expression of primary miR-9a (pri-miR-9a) and miR-9a precursor (pre-miR-9a). a Predicted stem–loop structure of pre-miR-9a. b Expression of miR-9a in S2 cells transfected with expression vectors including partial sequence of pri-miR-9a centered pre-miR-9a ( n = 6). c , d Expression levels of pri-miR-9a ( c , n = 8) and pre-miR-9a ( d , n = 8) in total cells after activation and inhibition of Dop1, respectively. e Expression levels of pri-miR-9a in the nucleus after activation and inhibition of Dop1, respectively. U6 was used as a nuclear marker and 18 S rRNA as a cytoplasmic marker. f , g Expression levels of pre-miR-9a in the nucleus ( f , n = 8) and cytoplasm ( g , n = 8) after activation and inhibition of Dop1, respectively. The asterisks outside the strip indicate the significant difference between controls and the treatments through Student’s t -test and presented as the mean ± SEM. ** P

    Techniques Used: Expressing, Transfection, Sequencing, Activation Assay, Inhibition, Marker, Stripping Membranes

    9) Product Images from "Evidence for an Essential Deglycosylation-Independent Activity of PNGase in Drosophila melanogaster"

    Article Title: Evidence for an Essential Deglycosylation-Independent Activity of PNGase in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010545

    Pngl also conserves characteristics of cytosolic PNGases from other species. (A) Schematic representation of PNGase orthologs from various species. Sc:  Saccharomyces cerevisiae ; At:  Arabidopsis thaliana ; Ce:  Caenorhabditis elegans ; Mm:  Mus musculus ; Dm:  Drosophila melanogaster ; the conserved core domain is colored by light grey. The transglutaminase domain with catalytic center: black, PUB domain: dotted-filled, and mannose-binding domain, filled with hatched lines. Two CXXC motifs are described with thin rectangles colored by dark grey. In the case of Dm, one of the rectangles is colored by white as CXXC is not conserved. (B) Cytosolic staining of (His) 6 -tagged Pngl expressed in  Drosophila  S2 cells, stained by anti-His antibody(red). Blue staining is for nuclear staining (DAPI) (C) Western blotting analysis of PNGase proteins expressed in Sf21 cells. Pngl was immunoprecipitaed with anti-Pngl antibody and was detected by Western blotting as described in “  Materials and Methods ”. Lane 1: Immunoprecipitated samples from mock-transfected Sf21 cell soluble fraction, lane 2: from Sf21 cells transfected with  Pngl , lane3: from larval disc-brain-complex soluble fraction. (D) Glycan binding assays of mouse (Ngly1) and fly (Pngl) PNGases. Lanes 1, 3, 5: negative control (no probe) of Ngly1, Pngl, and Pngl(C303A); lanes 2, 4, 6: Ngly1, Pngl, and Pngl(C303A) after reaction with the carbohydrate probe.
    Figure Legend Snippet: Pngl also conserves characteristics of cytosolic PNGases from other species. (A) Schematic representation of PNGase orthologs from various species. Sc: Saccharomyces cerevisiae ; At: Arabidopsis thaliana ; Ce: Caenorhabditis elegans ; Mm: Mus musculus ; Dm: Drosophila melanogaster ; the conserved core domain is colored by light grey. The transglutaminase domain with catalytic center: black, PUB domain: dotted-filled, and mannose-binding domain, filled with hatched lines. Two CXXC motifs are described with thin rectangles colored by dark grey. In the case of Dm, one of the rectangles is colored by white as CXXC is not conserved. (B) Cytosolic staining of (His) 6 -tagged Pngl expressed in Drosophila S2 cells, stained by anti-His antibody(red). Blue staining is for nuclear staining (DAPI) (C) Western blotting analysis of PNGase proteins expressed in Sf21 cells. Pngl was immunoprecipitaed with anti-Pngl antibody and was detected by Western blotting as described in “ Materials and Methods ”. Lane 1: Immunoprecipitated samples from mock-transfected Sf21 cell soluble fraction, lane 2: from Sf21 cells transfected with Pngl , lane3: from larval disc-brain-complex soluble fraction. (D) Glycan binding assays of mouse (Ngly1) and fly (Pngl) PNGases. Lanes 1, 3, 5: negative control (no probe) of Ngly1, Pngl, and Pngl(C303A); lanes 2, 4, 6: Ngly1, Pngl, and Pngl(C303A) after reaction with the carbohydrate probe.

    Techniques Used: Binding Assay, Staining, Western Blot, Immunoprecipitation, Transfection, Negative Control

    10) Product Images from "Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain"

    Article Title: Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200501113

    The Drosophila protein Croquemort and its mammalian paralogue CD36 are receptors for S. aureus . (a) Croquemort and the small GTPase Rac2 are specifically required for S. aureus binding and uptake. RNAi silencing of Croquemort (Crq) and Rac2 in Drosophila S2 cells reduced phagocytosis of S. aureus (left), but not E. coli (right). *, P ≤ 0.05; **, P ≤ 0.001; significantly different from control. (b and c) Expression of CD36 confers cellular binding and internalization of S. aureus and E. coli . (b) HEK293T-CD36 expressing cells (CD36; filled bars) demonstrate a threefold increase in binding of FITC-labeled S. aureus and a twofold increase in binding of FITC-labeled E. coli over vector-transfected cells (Mock; open bars). *, P ≤ 0.05. (c) Expression of CD36 by HEK293 cells confers a threefold increase in internalization of both FITC-labeled S. aureus and E. coli, as compared with vector-transfected cells (Mock; open bars). *, P ≤ 0.05.
    Figure Legend Snippet: The Drosophila protein Croquemort and its mammalian paralogue CD36 are receptors for S. aureus . (a) Croquemort and the small GTPase Rac2 are specifically required for S. aureus binding and uptake. RNAi silencing of Croquemort (Crq) and Rac2 in Drosophila S2 cells reduced phagocytosis of S. aureus (left), but not E. coli (right). *, P ≤ 0.05; **, P ≤ 0.001; significantly different from control. (b and c) Expression of CD36 confers cellular binding and internalization of S. aureus and E. coli . (b) HEK293T-CD36 expressing cells (CD36; filled bars) demonstrate a threefold increase in binding of FITC-labeled S. aureus and a twofold increase in binding of FITC-labeled E. coli over vector-transfected cells (Mock; open bars). *, P ≤ 0.05. (c) Expression of CD36 by HEK293 cells confers a threefold increase in internalization of both FITC-labeled S. aureus and E. coli, as compared with vector-transfected cells (Mock; open bars). *, P ≤ 0.05.

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

    11) Product Images from "Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling"

    Article Title: Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.09.011

    Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .
    Figure Legend Snippet: Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .

    Techniques Used: Inhibition, Radial Immuno Diffusion, Activation Assay, Recombinant, Quantitative RT-PCR, Expressing, Injection, Real-time Polymerase Chain Reaction

    PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p
    Figure Legend Snippet: PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p

    Techniques Used: Radial Immuno Diffusion, Expressing, Mutagenesis, Positive Control, Transfection, Fluorescence

    12) Product Images from "SH2B Regulation of Growth, Metabolism and Longevity in Both Insects and Mammals"

    Article Title: SH2B Regulation of Growth, Metabolism and Longevity in Both Insects and Mammals

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2010.04.002

    dSH2B directly promotes the IIS pathway (A–B) Adult flies (3 days) were randomly fed (F), fasted for 48 h (S), or re-fed for 24 h after 24 h starvation (R). The extracts were immunoblotted with the indicated antibodies. Akt phosphorylation was quantified by densitometry and normalized to total Akt protein levels (n=3). (C) Fat bodies were isolated from third instar larvae, and treated with or without human insulin (10 µg/ml) for 15 minutes. Cell extracts were immunoblotted with the indicated antibodies. (D) Fat body tissues were isolated from adult fly abdomens and treated with insulin (0.1, 1, and 10 µg/ml) as described in C. (E) S2 cells were transfected with dsRNAs against EGFP (si-EGFP) or dSH2B (si-dSH2B). dSH2B mRNA abundance was measured by quantitative RT-PCR 4 days after transfection (n=4). (F) S2 cells were cotransfected with dsRNAs and plasmids encoding V5-tagged dFOXO. Forty-eight h after transfection, cells were deprived of FBS for 6 h and treated with insulin (0.001, 0.01, 0.1, 1 and 10 µg/ml) for 15 min. Cells extracts were immunoblotted with the indicated antibodies. (G) V5-tagged dFOXO was transiently coexpressed with or without dSH2B in S2 cells, and treated with insulin (0.001, 0.01, 0.1, 1 and 10 µg/ml) as described in F. (H–I) S2 cells were cotransfected with dsRNAs and plasmids encoding V5-tagged dFOXO. Forty-eight h after transfection, cells were deprived of FBS for 6 h and treated with insulin (10 µg/ml) for 15 min. Cells were immunostained with anti-V5 antibody and visualized using a confocal microscopy. Cytoplasmic dFOXO-positive Cells were counted and normalized to the total number of cells (four independent experiments). *p
    Figure Legend Snippet: dSH2B directly promotes the IIS pathway (A–B) Adult flies (3 days) were randomly fed (F), fasted for 48 h (S), or re-fed for 24 h after 24 h starvation (R). The extracts were immunoblotted with the indicated antibodies. Akt phosphorylation was quantified by densitometry and normalized to total Akt protein levels (n=3). (C) Fat bodies were isolated from third instar larvae, and treated with or without human insulin (10 µg/ml) for 15 minutes. Cell extracts were immunoblotted with the indicated antibodies. (D) Fat body tissues were isolated from adult fly abdomens and treated with insulin (0.1, 1, and 10 µg/ml) as described in C. (E) S2 cells were transfected with dsRNAs against EGFP (si-EGFP) or dSH2B (si-dSH2B). dSH2B mRNA abundance was measured by quantitative RT-PCR 4 days after transfection (n=4). (F) S2 cells were cotransfected with dsRNAs and plasmids encoding V5-tagged dFOXO. Forty-eight h after transfection, cells were deprived of FBS for 6 h and treated with insulin (0.001, 0.01, 0.1, 1 and 10 µg/ml) for 15 min. Cells extracts were immunoblotted with the indicated antibodies. (G) V5-tagged dFOXO was transiently coexpressed with or without dSH2B in S2 cells, and treated with insulin (0.001, 0.01, 0.1, 1 and 10 µg/ml) as described in F. (H–I) S2 cells were cotransfected with dsRNAs and plasmids encoding V5-tagged dFOXO. Forty-eight h after transfection, cells were deprived of FBS for 6 h and treated with insulin (10 µg/ml) for 15 min. Cells were immunostained with anti-V5 antibody and visualized using a confocal microscopy. Cytoplasmic dFOXO-positive Cells were counted and normalized to the total number of cells (four independent experiments). *p

    Techniques Used: Isolation, Transfection, Quantitative RT-PCR, Confocal Microscopy

    dSH2B enhances the Chico pathway (A) S2 cells were transiently cotransfected with V5-tagged Chico plasmids and dSH2B plasmids. Cell extracts were prepared 48 h after transfection, immunoprecipitated with anti-V5 antibody, and immunoblotted with the indicated antibodies. Cell extracts were immunoblotted with anti-dSH2B or anti-V5 antibodies. (B) V5-tagged Chico was transiently co-expressed with or without dSH2B in S2 cells. Cells were treated with insulin for 15 minutes 48 h after transfection. Cell extracts were immunoprecipitated with anti-V5 antibody and immunoblotted with the indicated antibodies. (C) S2 cells were incubated with dsRNA for 4 days, transfected with plasmids encoding V5-tagged Chico, and treated with insulin for 15 minutes. Cell extracts were immunoprecipitated with anti-V5 antibody and immunoblotted with the indicated antibodies. (D) Chico mRNA abundance was measured by quantitative RT-PCR in wild type (WT) and Chico C/C (C/C) adult flies (32 flies per group). (E) Total TAG levels were measured in adult flies (3 days) and normalized to total protein levels (32 flies per group). (F) Survival curves of adult flies (3 days) in response to starvation (120 flies per group). *p
    Figure Legend Snippet: dSH2B enhances the Chico pathway (A) S2 cells were transiently cotransfected with V5-tagged Chico plasmids and dSH2B plasmids. Cell extracts were prepared 48 h after transfection, immunoprecipitated with anti-V5 antibody, and immunoblotted with the indicated antibodies. Cell extracts were immunoblotted with anti-dSH2B or anti-V5 antibodies. (B) V5-tagged Chico was transiently co-expressed with or without dSH2B in S2 cells. Cells were treated with insulin for 15 minutes 48 h after transfection. Cell extracts were immunoprecipitated with anti-V5 antibody and immunoblotted with the indicated antibodies. (C) S2 cells were incubated with dsRNA for 4 days, transfected with plasmids encoding V5-tagged Chico, and treated with insulin for 15 minutes. Cell extracts were immunoprecipitated with anti-V5 antibody and immunoblotted with the indicated antibodies. (D) Chico mRNA abundance was measured by quantitative RT-PCR in wild type (WT) and Chico C/C (C/C) adult flies (32 flies per group). (E) Total TAG levels were measured in adult flies (3 days) and normalized to total protein levels (32 flies per group). (F) Survival curves of adult flies (3 days) in response to starvation (120 flies per group). *p

    Techniques Used: Transfection, Immunoprecipitation, Incubation, Quantitative RT-PCR

    13) Product Images from "Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release"

    Article Title: Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200211087

    Rab5 and GFP-2xFYVE are associated to endosomal structures. Double labelings of Drosophila S2 cells showing (A) endogenous Rab5 immunostaining, (B) GFP-Rab5, or (C; left, green) GFP-2xFYVE and (A–C; middle, red) Texas red–dextran internalized upon a 5-min pulse. (Right panels) Overlay. Note that a substantial amount of the endogenous Rab5, GFP-Rab5, or GFP-2xFYVE–positive structures colocalize with the early endosomes where the dextran accumulates upon a short pulse. Bars, 10 μm.
    Figure Legend Snippet: Rab5 and GFP-2xFYVE are associated to endosomal structures. Double labelings of Drosophila S2 cells showing (A) endogenous Rab5 immunostaining, (B) GFP-Rab5, or (C; left, green) GFP-2xFYVE and (A–C; middle, red) Texas red–dextran internalized upon a 5-min pulse. (Right panels) Overlay. Note that a substantial amount of the endogenous Rab5, GFP-Rab5, or GFP-2xFYVE–positive structures colocalize with the early endosomes where the dextran accumulates upon a short pulse. Bars, 10 μm.

    Techniques Used: Immunostaining

    14) Product Images from "Cryo-electron microscopy structure of the lipid droplet-formation protein seipin"

    Article Title: Cryo-electron microscopy structure of the lipid droplet-formation protein seipin

    Journal: bioRxiv

    doi: 10.1101/418236

    The hydrophobic helix of the seipin ER luminal domain targets to LDs. (A) The molecular structure of the D. melanogaster hydrophobic helix highlighting residues spanning 172–192 in orange. (B) Helical plot of residues Leu175–Trp192. Non-polar residues are shown in yellow ( Gautier et al., 2008 ). Asterisks indicate residues mutated to Asp in the seipin-3D mutant (see below). (C) The helical region residue distribution for the top 200 seipin sequences (retrieved from Pfam database, corresponding to residues 175–192 of Drosophila seipin) shows evolutionary conservation of hydrophobicity ( Crooks et al., 2004 ). Residues are colored according to their physicochemical properties, with hydrophobic residues in orange. (D) The seipin hydrophobic helix binds artificial LDs in vitro . An Alexa488-labeled peptide comprising residues 174–193, but not a version with the 3D mutation (replacing Ile176, Ile176 and Trp182 with Asp), binds to artificial LDs. Scale bar, 20 µm. (E) Quantification of fluorescent signals from > 2000 artificial LDs per peptide as shown in C . (F) The seipin hydrophobic helix binds to the phospholipid monolayer in vitro . Seipin helix peptide, but not the mutated 3D version, preferentially binds to the phospholipid monolayer of TG lipid lenses incorporated into GUVs. A graphical representation, as well as representative confocal images show the peptide and phospholipid signals, respectively. Scale bar, 5 µm. (G) Quantification of enrichment on monolayer vs. bilayer of ≥18 GUVs per peptide as mean +/-standard deviation. (H) Binding of seipin hydrophobic helix to LDs in cells. The mCherry-tagged N-terminal amphipathic helical sequence (1–48), the luminal hydrophobic helix (174–193) of seipin, and a seipin-HH 3D mutant of the luminal helix were expressed in Drosophila S2 cells and analyzed by confocal imaging for LD binding. As a control, the CCT-M domain was expressed in S2 cells from the same vector. Scale bar, 5 µm. (I) Quantification of mCherry fluorescence on LDs vs. total signal per cell as shown in H as mean +/-standard deviation from ≥9 cells per construct.
    Figure Legend Snippet: The hydrophobic helix of the seipin ER luminal domain targets to LDs. (A) The molecular structure of the D. melanogaster hydrophobic helix highlighting residues spanning 172–192 in orange. (B) Helical plot of residues Leu175–Trp192. Non-polar residues are shown in yellow ( Gautier et al., 2008 ). Asterisks indicate residues mutated to Asp in the seipin-3D mutant (see below). (C) The helical region residue distribution for the top 200 seipin sequences (retrieved from Pfam database, corresponding to residues 175–192 of Drosophila seipin) shows evolutionary conservation of hydrophobicity ( Crooks et al., 2004 ). Residues are colored according to their physicochemical properties, with hydrophobic residues in orange. (D) The seipin hydrophobic helix binds artificial LDs in vitro . An Alexa488-labeled peptide comprising residues 174–193, but not a version with the 3D mutation (replacing Ile176, Ile176 and Trp182 with Asp), binds to artificial LDs. Scale bar, 20 µm. (E) Quantification of fluorescent signals from > 2000 artificial LDs per peptide as shown in C . (F) The seipin hydrophobic helix binds to the phospholipid monolayer in vitro . Seipin helix peptide, but not the mutated 3D version, preferentially binds to the phospholipid monolayer of TG lipid lenses incorporated into GUVs. A graphical representation, as well as representative confocal images show the peptide and phospholipid signals, respectively. Scale bar, 5 µm. (G) Quantification of enrichment on monolayer vs. bilayer of ≥18 GUVs per peptide as mean +/-standard deviation. (H) Binding of seipin hydrophobic helix to LDs in cells. The mCherry-tagged N-terminal amphipathic helical sequence (1–48), the luminal hydrophobic helix (174–193) of seipin, and a seipin-HH 3D mutant of the luminal helix were expressed in Drosophila S2 cells and analyzed by confocal imaging for LD binding. As a control, the CCT-M domain was expressed in S2 cells from the same vector. Scale bar, 5 µm. (I) Quantification of mCherry fluorescence on LDs vs. total signal per cell as shown in H as mean +/-standard deviation from ≥9 cells per construct.

    Techniques Used: Mutagenesis, In Vitro, Labeling, Standard Deviation, Binding Assay, Sequencing, Imaging, Plasmid Preparation, Fluorescence, Construct

    15) Product Images from "Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling"

    Article Title: Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling

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

    doi: 10.1073/pnas.1017548108

    Hipk phosphorylates Slimb to inhibit Arm ubiquitination without any effect on Arm phosphorylation. ( A ) In an in vitro kinase assay, Arm is sequentially phosphorylated in the presence of CK1 and Sgg (lane 2), as detected by a phospho-specific antibody against Arm (S44/S48). The presence of GST-Hipk has no effect on the phosphorylation of Arm by these kinases (lane 4). Phospho-Arm (S44/S48) is not detected in the absence of CK1 and Sgg (lanes 1 and 3). ( B ) In a pull-down assay, GST-Hipk forms a complex with Myc-Slimb extracted from Drosophila adults. The GST moiety alone does not bind Myc-Slimb. ( C ) In an in vitro kinase assay using radiolabeled ATP, immunoprecipitated Myc-Slimb is phosphorylated in the presence of GST-Hipk (lane 3) but not in its absence (lane 2). ( D ) In an in vitro ubiquitination assay, purified Arm is ubiquitinated in the presence of Slimb and other components of the ubiquitination machinery (lane 2) but not in the absence of Slimb (lane 1). Preincubation of GST-Hipk with Slimb in a kinase assay inhibits its ability to ubiquitinate Arm (lane 3). Preincubation of GST-Hipk with Slimb in the absence of ATP does not reduce Arm ubiquitination in the subsequent assay (lane 5). Preincubation of GST-Hipk with Arm in a kinase assay does not inhibit its Slimb-mediated ubiquitination (lane 6). ( E ) Protein lysates from Drosophila wing discs were assayed for levels of ubiquitinated Arm. Using omb-Gal4 to overexpress hipk results in lower levels of ubiquitinated Arm relative to wild-type discs. In the presence of the proteasome inhibitor MG132, wing discs reduced in function for hipk have higher levels of ubiquitinated Arm compared with wild-type discs. ( F ) In Drosophila S2 cells, Hipk enhances both the levels of cytosolic Arm (lane 2) and phospho-Arm (S44/S48) (lane 5), an effect that resembles the treatment of S2 cells with MG132 (lanes 3 and 6).
    Figure Legend Snippet: Hipk phosphorylates Slimb to inhibit Arm ubiquitination without any effect on Arm phosphorylation. ( A ) In an in vitro kinase assay, Arm is sequentially phosphorylated in the presence of CK1 and Sgg (lane 2), as detected by a phospho-specific antibody against Arm (S44/S48). The presence of GST-Hipk has no effect on the phosphorylation of Arm by these kinases (lane 4). Phospho-Arm (S44/S48) is not detected in the absence of CK1 and Sgg (lanes 1 and 3). ( B ) In a pull-down assay, GST-Hipk forms a complex with Myc-Slimb extracted from Drosophila adults. The GST moiety alone does not bind Myc-Slimb. ( C ) In an in vitro kinase assay using radiolabeled ATP, immunoprecipitated Myc-Slimb is phosphorylated in the presence of GST-Hipk (lane 3) but not in its absence (lane 2). ( D ) In an in vitro ubiquitination assay, purified Arm is ubiquitinated in the presence of Slimb and other components of the ubiquitination machinery (lane 2) but not in the absence of Slimb (lane 1). Preincubation of GST-Hipk with Slimb in a kinase assay inhibits its ability to ubiquitinate Arm (lane 3). Preincubation of GST-Hipk with Slimb in the absence of ATP does not reduce Arm ubiquitination in the subsequent assay (lane 5). Preincubation of GST-Hipk with Arm in a kinase assay does not inhibit its Slimb-mediated ubiquitination (lane 6). ( E ) Protein lysates from Drosophila wing discs were assayed for levels of ubiquitinated Arm. Using omb-Gal4 to overexpress hipk results in lower levels of ubiquitinated Arm relative to wild-type discs. In the presence of the proteasome inhibitor MG132, wing discs reduced in function for hipk have higher levels of ubiquitinated Arm compared with wild-type discs. ( F ) In Drosophila S2 cells, Hipk enhances both the levels of cytosolic Arm (lane 2) and phospho-Arm (S44/S48) (lane 5), an effect that resembles the treatment of S2 cells with MG132 (lanes 3 and 6).

    Techniques Used: In Vitro, Kinase Assay, Pull Down Assay, Immunoprecipitation, Ubiquitin Assay, Purification, Subsequent Assay

    Hipk2/Hipk inhibits SCF β-TrCP/Slimb -mediated ubiquitination of Gli3/Ci to promote Hedgehog signaling. ( A ) Transfection of COS7 cells with Hipk2 WT stabilizes endogenous, full-length Gli3 (lane 2), relative to the untransfected control (lane 1). Hipk2 K221R does not have any effect on the levels of full-length Gli3 (lane 3). ( B ) A transcriptional assay performed in Drosophila S2 cells shows that Hipk significantly up-regulates the expression of a Ci-responsive reporter gene ptcΔ136-Luc in a dose-dependent manner, both in the absence ( P
    Figure Legend Snippet: Hipk2/Hipk inhibits SCF β-TrCP/Slimb -mediated ubiquitination of Gli3/Ci to promote Hedgehog signaling. ( A ) Transfection of COS7 cells with Hipk2 WT stabilizes endogenous, full-length Gli3 (lane 2), relative to the untransfected control (lane 1). Hipk2 K221R does not have any effect on the levels of full-length Gli3 (lane 3). ( B ) A transcriptional assay performed in Drosophila S2 cells shows that Hipk significantly up-regulates the expression of a Ci-responsive reporter gene ptcΔ136-Luc in a dose-dependent manner, both in the absence ( P

    Techniques Used: Transfection, Transcription Factor Assay, Expressing

    16) Product Images from "Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion"

    Article Title: Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070327

    Accumulation of mouse and zebrafish PrPs at newly formed cell contacts in  Drosophila  S2 cells. A) Expression of the mouse PrP EGFP fusion construct (m PrP) induces cell contact formation and the subsequent accumulation of PrP at contact sites. This is not observed at fortuitous contacts formed by cells expressing a control construct lacking the major PrP domains (m PrP ΔCore). Cell-cell contacts are indicated by white arrowheads. Scale bars = 5 µm. B-D) Quantification of the number of S2 cell contacts showing accumulation of wild type (WT) and mutant constructs for mouse PrP (B), zebrafish PrP-1 (C) and zebrafish PrP-2 (D). Construct names are inserted in the graphs. Double and triple asterisks [** and ***] indicate statistical significance at  p
    Figure Legend Snippet: Accumulation of mouse and zebrafish PrPs at newly formed cell contacts in Drosophila S2 cells. A) Expression of the mouse PrP EGFP fusion construct (m PrP) induces cell contact formation and the subsequent accumulation of PrP at contact sites. This is not observed at fortuitous contacts formed by cells expressing a control construct lacking the major PrP domains (m PrP ΔCore). Cell-cell contacts are indicated by white arrowheads. Scale bars = 5 µm. B-D) Quantification of the number of S2 cell contacts showing accumulation of wild type (WT) and mutant constructs for mouse PrP (B), zebrafish PrP-1 (C) and zebrafish PrP-2 (D). Construct names are inserted in the graphs. Double and triple asterisks [** and ***] indicate statistical significance at p

    Techniques Used: Expressing, Construct, Mutagenesis

    17) Product Images from "Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells"

    Article Title: Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.22.7807-7816.2001

    Small RNA fragments generated by Drosophila S2 and mammalian cells. Cytoplasmic extracts (50 μg) from various mammalian cells were incubated with 30 nmol of radiolabeled dsRNA for 1 h at 30°C for S2 cells or 37°C for mammalian cells. After the reaction, samples were treated with proteinase K–0.5% SDS. The size of dsRNA was examined on a 12% denaturing acrylamide gel. Probe indicates the radiolabeled dsRNA made by in vitro transcription. Lane M, 10-bp markers.
    Figure Legend Snippet: Small RNA fragments generated by Drosophila S2 and mammalian cells. Cytoplasmic extracts (50 μg) from various mammalian cells were incubated with 30 nmol of radiolabeled dsRNA for 1 h at 30°C for S2 cells or 37°C for mammalian cells. After the reaction, samples were treated with proteinase K–0.5% SDS. The size of dsRNA was examined on a 12% denaturing acrylamide gel. Probe indicates the radiolabeled dsRNA made by in vitro transcription. Lane M, 10-bp markers.

    Techniques Used: Generated, Incubation, Acrylamide Gel Assay, In Vitro

    dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.
    Figure Legend Snippet: dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.

    Techniques Used: Produced, Sequencing, Inhibition, Expressing, In Situ, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, In Vitro, Staining

    18) Product Images from "Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex"

    Article Title: Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex

    Journal: Science signaling

    doi: 10.1126/scisignal.aar1976

    Lgl interacts with Vap33 in vitro and in vivo. ( A ) Protein interaction map summarizing the results of our proteomic data linking Lgl to Vap33 (red) and data from the DPiM linking the indicated proteins to Vap33 (blue). Proteins shown in yellow are subunits of the V-ATPase, and their known interactions are indicated in light gray. Proteins for which the human homolog interacts with the human Vap33 ortholog VAPA are outlined in green. ( B ) Representative immunoblot showing coimmunoprecipitation of Lgl-SBP and Vap33-V5 from S2 cells. Vap33-V5 was immunoprecipitated (IP) with an antibody recognizing V5 and immunoblotted (IB) with an antibody recognizing SBP. The experiment was performed twice. ( C  and  D ) Confocal planar images showing in situ PLA in third-instar larval eye discs. The positive PLA signals are punctate and shown in gray and magenta. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; green). Insets show high-magnification images of the PLA foci. (C) PLA on  MI07575 lgl-eGFP-lgl  eye discs using GFP and Vap33 antibodies. (D) PLA on  GMR > Vap33-HA  eye discs using HA and Lgl antibodies. The anterior region of the eye disc where the  GMR > Vap33-HA  is not expressed, and hence there is no HA epitope, acts as an internal negative control.  n  = 3 independent experiments,  n  = 3 or 4 samples per experiment. Scale bars, 50 µm.
    Figure Legend Snippet: Lgl interacts with Vap33 in vitro and in vivo. ( A ) Protein interaction map summarizing the results of our proteomic data linking Lgl to Vap33 (red) and data from the DPiM linking the indicated proteins to Vap33 (blue). Proteins shown in yellow are subunits of the V-ATPase, and their known interactions are indicated in light gray. Proteins for which the human homolog interacts with the human Vap33 ortholog VAPA are outlined in green. ( B ) Representative immunoblot showing coimmunoprecipitation of Lgl-SBP and Vap33-V5 from S2 cells. Vap33-V5 was immunoprecipitated (IP) with an antibody recognizing V5 and immunoblotted (IB) with an antibody recognizing SBP. The experiment was performed twice. ( C and D ) Confocal planar images showing in situ PLA in third-instar larval eye discs. The positive PLA signals are punctate and shown in gray and magenta. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; green). Insets show high-magnification images of the PLA foci. (C) PLA on MI07575 lgl-eGFP-lgl eye discs using GFP and Vap33 antibodies. (D) PLA on GMR > Vap33-HA eye discs using HA and Lgl antibodies. The anterior region of the eye disc where the GMR > Vap33-HA is not expressed, and hence there is no HA epitope, acts as an internal negative control. n = 3 independent experiments, n = 3 or 4 samples per experiment. Scale bars, 50 µm.

    Techniques Used: In Vitro, In Vivo, Immunoprecipitation, In Situ, Proximity Ligation Assay, Staining, Negative Control

    Lgl facilitates Vap33 interaction with the V-ATPase component Vha68-3. ( A ) Representative immunoblot showing coimmunoprecipitation of Vha68–3–HA and Vap33-V5 from S2 cells. Vap33-V5 was immunoprecipitated (IP) with an antibody specific to V5 and immunoblotted (IB) with an antibody specific for HA. ( B ) Representative immunoblot showing the abundance of Lgl-SBP in S2 cells in which Lgl or Bla (control) was knocked down by RNAi. ( C ) Representative immunoblot showing coimmunoprecipitation of Vha68–3–HA and Vap33-V5 from extracts of S2 cells in which Lgl was knocked down. Vap33-V5 was immunoprecipitated with an antibody specific for V5 and blotted with an antibody specific for HA. The experiments in (A) to (C) were conducted twice. ( D ) Confocal planar images showing the PLA for Vha44-GFP and Vap33 in the posterior region of third-instar larval lgl 27S3 mosaic eye discs. Mutant tissue is marked by the absence of RFP in the posterior of the eye disc, using GMR-RFP as a clonal marker in eye discs expressing the Vha44 protein trap Vha44-GFP throughout the tissue. The positive PLA signal is punctate and shown in gray (left) or green (right). Scale bar, 10 µm. ( E ) Quantification of the PLA foci in lgl 27S3 mutant tissue compared to WT tissue, plotted as average density per megapixel. n = 3 independent experiments, n = 4 samples analyzed per genotype per experiment. Error bars indicate SEM. ** P = 0.005 ( t tests with two-tailed distribution and unequal variance). ( F ) Model for the regulation of V-ATPase activity by Lgl through Vap33. Lgl binds to and activates or stabilizes Vap33, which, in turn, binds to V-ATPase subunits and inhibits V-ATPase activity, thereby reducing γ-secretase activity and Notch activation.
    Figure Legend Snippet: Lgl facilitates Vap33 interaction with the V-ATPase component Vha68-3. ( A ) Representative immunoblot showing coimmunoprecipitation of Vha68–3–HA and Vap33-V5 from S2 cells. Vap33-V5 was immunoprecipitated (IP) with an antibody specific to V5 and immunoblotted (IB) with an antibody specific for HA. ( B ) Representative immunoblot showing the abundance of Lgl-SBP in S2 cells in which Lgl or Bla (control) was knocked down by RNAi. ( C ) Representative immunoblot showing coimmunoprecipitation of Vha68–3–HA and Vap33-V5 from extracts of S2 cells in which Lgl was knocked down. Vap33-V5 was immunoprecipitated with an antibody specific for V5 and blotted with an antibody specific for HA. The experiments in (A) to (C) were conducted twice. ( D ) Confocal planar images showing the PLA for Vha44-GFP and Vap33 in the posterior region of third-instar larval lgl 27S3 mosaic eye discs. Mutant tissue is marked by the absence of RFP in the posterior of the eye disc, using GMR-RFP as a clonal marker in eye discs expressing the Vha44 protein trap Vha44-GFP throughout the tissue. The positive PLA signal is punctate and shown in gray (left) or green (right). Scale bar, 10 µm. ( E ) Quantification of the PLA foci in lgl 27S3 mutant tissue compared to WT tissue, plotted as average density per megapixel. n = 3 independent experiments, n = 4 samples analyzed per genotype per experiment. Error bars indicate SEM. ** P = 0.005 ( t tests with two-tailed distribution and unequal variance). ( F ) Model for the regulation of V-ATPase activity by Lgl through Vap33. Lgl binds to and activates or stabilizes Vap33, which, in turn, binds to V-ATPase subunits and inhibits V-ATPase activity, thereby reducing γ-secretase activity and Notch activation.

    Techniques Used: Immunoprecipitation, Proximity Ligation Assay, Mutagenesis, Marker, Expressing, Two Tailed Test, Activity Assay, Activation Assay

    19) Product Images from "Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)"

    Article Title: Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072592

    Activation of the promoters of Drosophila and shrimp AMP genes by overexpression of LvIAP2 in Drosophila S2 cells. Drosophila S2 cells were transfected with a protein expression vector (pAC5.1 empty vector or pAC5.1-LvIAP2 vector), a luciferase reporter plasmid (pGL3-Basic, pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs, or pGL3-AttA), and pRL-TK Renilla luciferase plasmid (as an internal control) (Promega, USA). Thirty-six hours later, the cells were harvested for examination of luciferase activities using the dual luciferase reporter assay system (Promega, USA). All data are representative of three independent experiments. The bars indicate the mean ± S.D. of the luciferase activity (n = 3).
    Figure Legend Snippet: Activation of the promoters of Drosophila and shrimp AMP genes by overexpression of LvIAP2 in Drosophila S2 cells. Drosophila S2 cells were transfected with a protein expression vector (pAC5.1 empty vector or pAC5.1-LvIAP2 vector), a luciferase reporter plasmid (pGL3-Basic, pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs, or pGL3-AttA), and pRL-TK Renilla luciferase plasmid (as an internal control) (Promega, USA). Thirty-six hours later, the cells were harvested for examination of luciferase activities using the dual luciferase reporter assay system (Promega, USA). All data are representative of three independent experiments. The bars indicate the mean ± S.D. of the luciferase activity (n = 3).

    Techniques Used: Activation Assay, Over Expression, Transfection, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

    Subcellular localization of LvIAP1 (A), LvIAP2 (B), and LvIAP3 (C) in Drosophila S2 cells. Drosophila S2 cells were transfected with the pAc5.1-LvIAP1-3-GFP plasmids. At 36 hours post-transfection, the cover slips were washed, fixed, and stained with Hoechst 33258. The protein cellular localization was examined under a Leica laser scanning confocal microscope. The nuclei were visualized using the Hoechst stain (blue).
    Figure Legend Snippet: Subcellular localization of LvIAP1 (A), LvIAP2 (B), and LvIAP3 (C) in Drosophila S2 cells. Drosophila S2 cells were transfected with the pAc5.1-LvIAP1-3-GFP plasmids. At 36 hours post-transfection, the cover slips were washed, fixed, and stained with Hoechst 33258. The protein cellular localization was examined under a Leica laser scanning confocal microscope. The nuclei were visualized using the Hoechst stain (blue).

    Techniques Used: Transfection, Staining, Microscopy

    Activation of the promoters of  WSSV069  ( ie1 ),  WSSV303 , and  WSSV371  by overexpression of LvIAP2 in  Drosophila  S2 cells.
    Figure Legend Snippet: Activation of the promoters of WSSV069 ( ie1 ), WSSV303 , and WSSV371 by overexpression of LvIAP2 in Drosophila S2 cells.

    Techniques Used: Activation Assay, Over Expression

    20) Product Images from "Drosophila Schneider 2 (S2) cells: A novel tool for studying HSV-induced membrane fusion"

    Article Title: Drosophila Schneider 2 (S2) cells: A novel tool for studying HSV-induced membrane fusion

    Journal: Virology

    doi: 10.1016/j.virol.2013.01.004

    PILRα-Ig did not bind to gB in S2 cells by immunoprecipitation and monolayer binding assay
    Figure Legend Snippet: PILRα-Ig did not bind to gB in S2 cells by immunoprecipitation and monolayer binding assay

    Techniques Used: Immunoprecipitation, Binding Assay

    Infection of Vero cells and S2 cells by HSV-1
    Figure Legend Snippet: Infection of Vero cells and S2 cells by HSV-1

    Techniques Used: Infection

    Expression of FLAG-tagged receptors and glycoproteins in S2 cells
    Figure Legend Snippet: Expression of FLAG-tagged receptors and glycoproteins in S2 cells

    Techniques Used: Expressing

    Absence of the CHO-K1 endogenous receptor in S2 cells
    Figure Legend Snippet: Absence of the CHO-K1 endogenous receptor in S2 cells

    Techniques Used:

    Mobility of gB and PILRα in S2 cells as well as Neuraminidase treatment
    Figure Legend Snippet: Mobility of gB and PILRα in S2 cells as well as Neuraminidase treatment

    Techniques Used:

    21) Product Images from "Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human Glutamate Carboxypeptidase II"

    Article Title: Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human Glutamate Carboxypeptidase II

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2011.11.016

    Schematic representation of Avi-GCPII and analysis of its purification Panel A: Schematic representation of Avi-GCPII with amino acid sequences detailed for all AviTEV tag parts; Avi – sequence of 15 amino acids known as AviTag, which is specifically recognized by biotin-protein ligase and biotinylated on the ε-amino group of the lysine residue (underlined); TEV – protein sequence specifically recognized by TEV protease (cleavage site is marked with *); rhGCPII - extracellular portion of GCPII consisting of amino acids 44–750. ▼ - identified cleavage site within Avi-GCPII recognized by an unknown host protease which is co-purified with Avi-GCPII. Spacer sequence and amino acids introduced during molecular cloning are depicted in a smaller font size. Panel B: Analysis of Avi-GCPII purification on Streptavidin Mutein Matrix. Equilibrated concentrated conditioned medium from S2 cells was mixed with resin and incubated overnight at 4°C. The resin was separated from the medium on a gravity-flow column, and purified protein was eluted with an excess of D-biotin. Individual fractions obtained during purification were subsequently analyzed by SDS-PAGE and stained by Coomassie blue. 1. Molecular weight marker; 2. Load; 3. Flow-through; 4. Wash; 5–11. Elutions 1–7. 4 µl of the sample was loaded to each lane. Detailed descriptions of individual fractions can be found in Materials and Methods.
    Figure Legend Snippet: Schematic representation of Avi-GCPII and analysis of its purification Panel A: Schematic representation of Avi-GCPII with amino acid sequences detailed for all AviTEV tag parts; Avi – sequence of 15 amino acids known as AviTag, which is specifically recognized by biotin-protein ligase and biotinylated on the ε-amino group of the lysine residue (underlined); TEV – protein sequence specifically recognized by TEV protease (cleavage site is marked with *); rhGCPII - extracellular portion of GCPII consisting of amino acids 44–750. ▼ - identified cleavage site within Avi-GCPII recognized by an unknown host protease which is co-purified with Avi-GCPII. Spacer sequence and amino acids introduced during molecular cloning are depicted in a smaller font size. Panel B: Analysis of Avi-GCPII purification on Streptavidin Mutein Matrix. Equilibrated concentrated conditioned medium from S2 cells was mixed with resin and incubated overnight at 4°C. The resin was separated from the medium on a gravity-flow column, and purified protein was eluted with an excess of D-biotin. Individual fractions obtained during purification were subsequently analyzed by SDS-PAGE and stained by Coomassie blue. 1. Molecular weight marker; 2. Load; 3. Flow-through; 4. Wash; 5–11. Elutions 1–7. 4 µl of the sample was loaded to each lane. Detailed descriptions of individual fractions can be found in Materials and Methods.

    Techniques Used: Purification, Sequencing, Molecular Cloning, Incubation, Flow Cytometry, SDS Page, Staining, Molecular Weight, Marker

    22) Product Images from "ErbB Inhibitory Protein [EBIP]: A Modified Ectodomain of EGFR Synergizes with Dasatinib to Inhibit Growth of Breast Cancer Cells"

    Article Title: ErbB Inhibitory Protein [EBIP]: A Modified Ectodomain of EGFR Synergizes with Dasatinib to Inhibit Growth of Breast Cancer Cells

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-10-0019

    (A) Schematic representation of full length human EGFR and four different plamid constructs of (i) Rat ERRP 1-478, (ii) Rat EGFR ectodomain [ERRP without “U” region; referred to as ERRP-447], (iii) Human EGFR ectodomain (referred to as hEGFR-501), and (iv) Human EGFR ectodomain fused with “U” region [referred to as hEGFR-448+U or EBIP]. (B) Synthesis of recombinant proteins by drosophila S2 cells in the absence (-) or presence (+) of CuSO4 as determined by western-blot analysis of the cell lysates. Recombinant proteins containing both His- and V5- tags, were purified using His-tag and immuno-blotted with V5- anti-body. (C) Western blot analysis of EBIP localization in response to TGF-α induction of breast cancer cells. After 8 hours of incubation with EBIP, MDA-MB-468 cells, which were serum-starved, were induced with TGF- α. The cell lysates were immunoprecipitated with EGFR antibodies overnight and the immunoprecipitates were subsequently subjected to Western blot analysis with V5 antibody for EBIP detection. (D) Inhibition of growth of MDA-MB-468 cells in response to immuno-affinity purified EBIP and ERRP.
    Figure Legend Snippet: (A) Schematic representation of full length human EGFR and four different plamid constructs of (i) Rat ERRP 1-478, (ii) Rat EGFR ectodomain [ERRP without “U” region; referred to as ERRP-447], (iii) Human EGFR ectodomain (referred to as hEGFR-501), and (iv) Human EGFR ectodomain fused with “U” region [referred to as hEGFR-448+U or EBIP]. (B) Synthesis of recombinant proteins by drosophila S2 cells in the absence (-) or presence (+) of CuSO4 as determined by western-blot analysis of the cell lysates. Recombinant proteins containing both His- and V5- tags, were purified using His-tag and immuno-blotted with V5- anti-body. (C) Western blot analysis of EBIP localization in response to TGF-α induction of breast cancer cells. After 8 hours of incubation with EBIP, MDA-MB-468 cells, which were serum-starved, were induced with TGF- α. The cell lysates were immunoprecipitated with EGFR antibodies overnight and the immunoprecipitates were subsequently subjected to Western blot analysis with V5 antibody for EBIP detection. (D) Inhibition of growth of MDA-MB-468 cells in response to immuno-affinity purified EBIP and ERRP.

    Techniques Used: Construct, Recombinant, Western Blot, Purification, Incubation, Multiple Displacement Amplification, Immunoprecipitation, Inhibition, Affinity Purification

    23) Product Images from "In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation"

    Article Title: In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation

    Journal: eLife

    doi: 10.7554/eLife.21475

    PAD in cellular stress. ( A ) Schematics of YFP-PAD probe. ( B ) YFP-PAD in growing (Schneider’s) and amino-acid starved (KRB) cells for 3 hr. Note that with the exception of an increase of the nuclear intensity, amino-acid starvation does not lead to the formation of a cytoplasmic pattern. ( C ) YFP-PAD in S2 cells upon arsenate treatment. Note the formation of a robust YFP-PAD cytoplasmic pattern that co-localises with stress granules (FMR1, red). Scale bars: 10 μm DOI: http://dx.doi.org/10.7554/eLife.21475.002 10.7554/eLife.21475.003 List of the primers used in this manuscript. DOI: http://dx.doi.org/10.7554/eLife.21475.003
    Figure Legend Snippet: PAD in cellular stress. ( A ) Schematics of YFP-PAD probe. ( B ) YFP-PAD in growing (Schneider’s) and amino-acid starved (KRB) cells for 3 hr. Note that with the exception of an increase of the nuclear intensity, amino-acid starvation does not lead to the formation of a cytoplasmic pattern. ( C ) YFP-PAD in S2 cells upon arsenate treatment. Note the formation of a robust YFP-PAD cytoplasmic pattern that co-localises with stress granules (FMR1, red). Scale bars: 10 μm DOI: http://dx.doi.org/10.7554/eLife.21475.002 10.7554/eLife.21475.003 List of the primers used in this manuscript. DOI: http://dx.doi.org/10.7554/eLife.21475.003

    Techniques Used:

    dPARP16 is anchored at the ER. IF visualisation of V5-dPARP16 (green) with respect to the ER marker Calnexin (red) after 1 hr expression in growing S2 cells (Schneider’s). Although PARP16 only partially co-localises with calnexin, the ER localisation was confirmed by IEM (red circles). N; Nucleus. Scale bar: 10 μm (left) and 200 nm (right). DOI: http://dx.doi.org/10.7554/eLife.21475.011
    Figure Legend Snippet: dPARP16 is anchored at the ER. IF visualisation of V5-dPARP16 (green) with respect to the ER marker Calnexin (red) after 1 hr expression in growing S2 cells (Schneider’s). Although PARP16 only partially co-localises with calnexin, the ER localisation was confirmed by IEM (red circles). N; Nucleus. Scale bar: 10 μm (left) and 200 nm (right). DOI: http://dx.doi.org/10.7554/eLife.21475.011

    Techniques Used: Marker, Expressing

    dPARP16 catalytic activity and membrane anchoring is required for Sec body formation. ( A, A’ ) IF visualisation of Sec body formation (Sec16, red) in growing S2 cells (Schneider’s) expressing wild type and catalytic mutant dPARP16 (Y199A and Y221A) (green) ( B ). Note that the expression of the catalytic mutants does not drive Sec body formation (arrowhead in B ) whereas the wild type dPARP16 does (arrow in A ) (quantified in A’ ). ( B, B’ ) IF visualisation of Sec body formation (Sec16, red) in amino-acid starved S2 cells depleted of dPARP16 (3’UTR) and expressing wild type dPARP16 and Y199A dPARP16 catalytic mutant ( B ). Note that the mutant does not rescue Sec body formation (arrowhead in C ) whereas the wild type dPARP16 does (arrow in B) (quantified in B’ ). ( C ) IF visualisation of Sec16 (red) upon wild type V5-dPARP16 and ΔTM V5-dPARP16 expression (green) for 3 hr in Schneider’s. Note that overexpressed wild type dPARP16 forms rings and spots (green arrows) as well as Sec bodies (white arrows) whereas the ΔTM V5 dPARP16 does not (arrowheads) (quantified in A’ ). ( D ) IF visualisation in confocal sections of V5-dPARP16 and dPARP16-GFP (green) and Sec16 (red) after 1 hr incubation in KRB. Note that the forming Sec bodies localise closely to dPARP16. Scale bars: 10 μm. Error bars: SEM. DOI: http://dx.doi.org/10.7554/eLife.21475.016
    Figure Legend Snippet: dPARP16 catalytic activity and membrane anchoring is required for Sec body formation. ( A, A’ ) IF visualisation of Sec body formation (Sec16, red) in growing S2 cells (Schneider’s) expressing wild type and catalytic mutant dPARP16 (Y199A and Y221A) (green) ( B ). Note that the expression of the catalytic mutants does not drive Sec body formation (arrowhead in B ) whereas the wild type dPARP16 does (arrow in A ) (quantified in A’ ). ( B, B’ ) IF visualisation of Sec body formation (Sec16, red) in amino-acid starved S2 cells depleted of dPARP16 (3’UTR) and expressing wild type dPARP16 and Y199A dPARP16 catalytic mutant ( B ). Note that the mutant does not rescue Sec body formation (arrowhead in C ) whereas the wild type dPARP16 does (arrow in B) (quantified in B’ ). ( C ) IF visualisation of Sec16 (red) upon wild type V5-dPARP16 and ΔTM V5-dPARP16 expression (green) for 3 hr in Schneider’s. Note that overexpressed wild type dPARP16 forms rings and spots (green arrows) as well as Sec bodies (white arrows) whereas the ΔTM V5 dPARP16 does not (arrowheads) (quantified in A’ ). ( D ) IF visualisation in confocal sections of V5-dPARP16 and dPARP16-GFP (green) and Sec16 (red) after 1 hr incubation in KRB. Note that the forming Sec bodies localise closely to dPARP16. Scale bars: 10 μm. Error bars: SEM. DOI: http://dx.doi.org/10.7554/eLife.21475.016

    Techniques Used: Activity Assay, Size-exclusion Chromatography, Expressing, Mutagenesis, Incubation

    GFP-MAD design and optimisation. Schematics representation of several version of GFP-MAD based upon macrodomains 1–3 of PARP14. Note that the macrodomains of human PARP14 are more efficient at detecting MARylation in S2 cells than those from mouse PARP14 that were originally used by ( Forst et al., 2013 ). Furthermore, the insertion of a linker greatly improved the probe sensitivity and/or efficiency. DOI: http://dx.doi.org/10.7554/eLife.21475.005
    Figure Legend Snippet: GFP-MAD design and optimisation. Schematics representation of several version of GFP-MAD based upon macrodomains 1–3 of PARP14. Note that the macrodomains of human PARP14 are more efficient at detecting MARylation in S2 cells than those from mouse PARP14 that were originally used by ( Forst et al., 2013 ). Furthermore, the insertion of a linker greatly improved the probe sensitivity and/or efficiency. DOI: http://dx.doi.org/10.7554/eLife.21475.005

    Techniques Used:

    Screen for PARPs in MAD spot formation. IF visualisation of GFP-MAD spots in amino-acid starved (KRB) mock, dPARP1, dTNK and dPARP16 depleted S2 cells. Note that, with exception of dPARP16 depletion, GFP-MAD spots formation is not affected by dPARP1 and dTNK depletions. Scale bar: 10 μm DOI: http://dx.doi.org/10.7554/eLife.21475.008
    Figure Legend Snippet: Screen for PARPs in MAD spot formation. IF visualisation of GFP-MAD spots in amino-acid starved (KRB) mock, dPARP1, dTNK and dPARP16 depleted S2 cells. Note that, with exception of dPARP16 depletion, GFP-MAD spots formation is not affected by dPARP1 and dTNK depletions. Scale bar: 10 μm DOI: http://dx.doi.org/10.7554/eLife.21475.008

    Techniques Used:

    dPARP1 and dTNK depletion does not affect Sec body formation upon amino-acid starvation. ( A, B ) IF visualisation of endogenous Sec16 (red) in amino-acid starved (KRB) S2 cells that are mock, dPARP1 and dTNK depleted. Note that Sec body formation is not affected as they form as efficiently in all conditions (quantified in B ). Scale bars: 10 μm. Error bars: SEM. DOI: http://dx.doi.org/10.7554/eLife.21475.014
    Figure Legend Snippet: dPARP1 and dTNK depletion does not affect Sec body formation upon amino-acid starvation. ( A, B ) IF visualisation of endogenous Sec16 (red) in amino-acid starved (KRB) S2 cells that are mock, dPARP1 and dTNK depleted. Note that Sec body formation is not affected as they form as efficiently in all conditions (quantified in B ). Scale bars: 10 μm. Error bars: SEM. DOI: http://dx.doi.org/10.7554/eLife.21475.014

    Techniques Used: Size-exclusion Chromatography

    dPARP1 and dTNK overexpression does not lead to Sec body formation in growing conditions. IF visualisation of endogenous Sec16 (red) in S2 cells expressing dPARP1 and dTNK in Schneider’s and KRB. Note that the overexpression of any of these enzymes neither leads to Sec body formation in Schneider’s nor affects their formation in KRB. Scale bars: 10 μm. Of note: dTNK, a cytoplasmic predicted MARylation enzyme, co-localises robustly with ERES in S2 cells, both under growing conditions and upon amino-acid starvation. Furthermore, its overexpression leads to the remodelling of the ERES. Yet, neither its overexpression nor its depletion has an effect on Sec body formation. This shows that the localization of MARylation enzymes at/near ERES is not sufficient to displace Sec16 and elicit a stress responsedARDT15 MARylation activity is substrate (and stress) specific. DOI: http://dx.doi.org/10.7554/eLife.21475.015
    Figure Legend Snippet: dPARP1 and dTNK overexpression does not lead to Sec body formation in growing conditions. IF visualisation of endogenous Sec16 (red) in S2 cells expressing dPARP1 and dTNK in Schneider’s and KRB. Note that the overexpression of any of these enzymes neither leads to Sec body formation in Schneider’s nor affects their formation in KRB. Scale bars: 10 μm. Of note: dTNK, a cytoplasmic predicted MARylation enzyme, co-localises robustly with ERES in S2 cells, both under growing conditions and upon amino-acid starvation. Furthermore, its overexpression leads to the remodelling of the ERES. Yet, neither its overexpression nor its depletion has an effect on Sec body formation. This shows that the localization of MARylation enzymes at/near ERES is not sufficient to displace Sec16 and elicit a stress responsedARDT15 MARylation activity is substrate (and stress) specific. DOI: http://dx.doi.org/10.7554/eLife.21475.015

    Techniques Used: Over Expression, Size-exclusion Chromatography, Expressing, Activity Assay

    24) Product Images from "SR proteins control a complex network of RNA-processing events"

    Article Title: SR proteins control a complex network of RNA-processing events

    Journal: RNA

    doi: 10.1261/rna.043893.113

    Genome-wide analysis of SR-dependent AS events. Drosophila melanogaster S2 cells were treated with SR-RNAi. ( A ) Western blot analysis of S2 cells treated with two nonoverlapping SR-specific dsRNAs versus S2 cells treated with nonspecific dsRNA (GFP); loading control, α-tubulin. ( B ) RNA-seq analysis showing the total number of simple AS events regulated by each SR protein; each bar shows the proportion of events that are less included (SR-activated, red) or more included (SR-repressed, dark gray). ( C ) RT-PCR validation of putative SR-regulated AS events identified by RNA-seq analysis (significantly changed events [*] P ≤ 0.05, [**] P ≤ 0.01, [***] P ≤ 0.001 Fisher's exact test); events separated into activator, repressor, or antagonistic. PSI values calculated from the RT-PCR and RNA-seq experiments are shown for comparison below each lane. Gene model is displayed below each gel with arrows indicating primer locations for PCR of the alternative region (red). ( D ) Heatmap reveals hierarchical clustering of PSI-switch scores for SR-regulated AS events ( y -axis) for each SR protein ( x -axis).
    Figure Legend Snippet: Genome-wide analysis of SR-dependent AS events. Drosophila melanogaster S2 cells were treated with SR-RNAi. ( A ) Western blot analysis of S2 cells treated with two nonoverlapping SR-specific dsRNAs versus S2 cells treated with nonspecific dsRNA (GFP); loading control, α-tubulin. ( B ) RNA-seq analysis showing the total number of simple AS events regulated by each SR protein; each bar shows the proportion of events that are less included (SR-activated, red) or more included (SR-repressed, dark gray). ( C ) RT-PCR validation of putative SR-regulated AS events identified by RNA-seq analysis (significantly changed events [*] P ≤ 0.05, [**] P ≤ 0.01, [***] P ≤ 0.001 Fisher's exact test); events separated into activator, repressor, or antagonistic. PSI values calculated from the RT-PCR and RNA-seq experiments are shown for comparison below each lane. Gene model is displayed below each gel with arrows indicating primer locations for PCR of the alternative region (red). ( D ) Heatmap reveals hierarchical clustering of PSI-switch scores for SR-regulated AS events ( y -axis) for each SR protein ( x -axis).

    Techniques Used: Genome Wide, Western Blot, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Combinatorial regulation of AS by SR proteins. Drosophila melanogaster S2 cells were treated with RNAi against one or more of the eight SR proteins and RNA-seq was performed to detect the changes in AS. ( A ) Proportion of AS events regulated by one (dark gray) or more than one (red) SR protein. ( B ) Heatmap of coregulated AS events identified by RNA-seq subdivided by those with increased inclusion or exclusion. ( C ) Western blot analysis of lysates from S2 cells treated with dsRNA against one or two SR proteins and dsRNA against GFP as a control; α-tubulin used as a loading control. ( D–F ) S2 cells were treated with RNAi against one SR protein (SC35, B52 or XL6) or two (SC35 and B52 or XL6 and B52) and RNA isolated. RNA-seq was used to create a heatmap showing hierarchical clustering of PSI-switch scores for the resulting changes in AS events ( D ), graph displaying the total number of significantly changed events in each SR protein-depleted RNA-seq experiment ( E ), and number of coregulated events that displayed a greater (enhanced; red), equal or less (cooperative; blue), or opposite (compensatory; green) PSI change in the simultaneous knockdown compared with individual SR knockdown ( F ). ( G ) RT-PCR of coregulated AS events. PSI values and mean ΔPSI values from control displayed below each lane. Gene model is displayed below each gel with arrows indicating primer locations for PCR of the alternative region (red).
    Figure Legend Snippet: Combinatorial regulation of AS by SR proteins. Drosophila melanogaster S2 cells were treated with RNAi against one or more of the eight SR proteins and RNA-seq was performed to detect the changes in AS. ( A ) Proportion of AS events regulated by one (dark gray) or more than one (red) SR protein. ( B ) Heatmap of coregulated AS events identified by RNA-seq subdivided by those with increased inclusion or exclusion. ( C ) Western blot analysis of lysates from S2 cells treated with dsRNA against one or two SR proteins and dsRNA against GFP as a control; α-tubulin used as a loading control. ( D–F ) S2 cells were treated with RNAi against one SR protein (SC35, B52 or XL6) or two (SC35 and B52 or XL6 and B52) and RNA isolated. RNA-seq was used to create a heatmap showing hierarchical clustering of PSI-switch scores for the resulting changes in AS events ( D ), graph displaying the total number of significantly changed events in each SR protein-depleted RNA-seq experiment ( E ), and number of coregulated events that displayed a greater (enhanced; red), equal or less (cooperative; blue), or opposite (compensatory; green) PSI change in the simultaneous knockdown compared with individual SR knockdown ( F ). ( G ) RT-PCR of coregulated AS events. PSI values and mean ΔPSI values from control displayed below each lane. Gene model is displayed below each gel with arrows indicating primer locations for PCR of the alternative region (red).

    Techniques Used: RNA Sequencing Assay, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    SR protein regulation of AS depends on RRM-domain and requires RS-domain. ( A ) Schematic showing relevant domains of three copper-inducible proteins—all tagged with 2x Flag and 2x HA: RRM, RNA-binding; Zn, zinc knuckle; RS, C-terminal arginine/serine. ( B ) Western blot of endogenous and recombinant XL6 in Drosophila S2 cells assayed in the presence or absence of dsRNA against endogenous XL6 and induction or recombinant XL6 isoforms; loading control, histone H3. ( C ) Semiquantitative RT-PCR of XL6-regulated targets ( Dre4 , Vps35 , DppIII ) and B52-regulated controls ( CG12065 and Syb ) in transfected S2 cells expressing wild-type or mutant XL6 (Expressed) and cultured in the presence or absence of dsRNA against endogenous XL6 (XL6 RNAi). Transcript models showing exon and intron structure (exon, thick box; intron, thin line; alternative region, red). PSI values are displayed below each lane. ( D ) Semiquantitative RT-PCR of AS targets in untransfected S2 cells cultured in the presence of dsRNA to either nonspecific sequence (control) or B52. PSI values are displayed below each lane.
    Figure Legend Snippet: SR protein regulation of AS depends on RRM-domain and requires RS-domain. ( A ) Schematic showing relevant domains of three copper-inducible proteins—all tagged with 2x Flag and 2x HA: RRM, RNA-binding; Zn, zinc knuckle; RS, C-terminal arginine/serine. ( B ) Western blot of endogenous and recombinant XL6 in Drosophila S2 cells assayed in the presence or absence of dsRNA against endogenous XL6 and induction or recombinant XL6 isoforms; loading control, histone H3. ( C ) Semiquantitative RT-PCR of XL6-regulated targets ( Dre4 , Vps35 , DppIII ) and B52-regulated controls ( CG12065 and Syb ) in transfected S2 cells expressing wild-type or mutant XL6 (Expressed) and cultured in the presence or absence of dsRNA against endogenous XL6 (XL6 RNAi). Transcript models showing exon and intron structure (exon, thick box; intron, thin line; alternative region, red). PSI values are displayed below each lane. ( D ) Semiquantitative RT-PCR of AS targets in untransfected S2 cells cultured in the presence of dsRNA to either nonspecific sequence (control) or B52. PSI values are displayed below each lane.

    Techniques Used: RNA Binding Assay, Western Blot, Recombinant, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Mutagenesis, Cell Culture, Sequencing

    25) Product Images from "REPTOR and REPTOR-BP regulate organismal metabolism and transcription downstream of TORC1"

    Article Title: REPTOR and REPTOR-BP regulate organismal metabolism and transcription downstream of TORC1

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2015.03.013

    REPTOR and REPTOR-BP are required for activation of almost all rapamycin-induced genes in S2 cells
    Figure Legend Snippet: REPTOR and REPTOR-BP are required for activation of almost all rapamycin-induced genes in S2 cells

    Techniques Used: Activation Assay

    26) Product Images from "Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1) *"

    Article Title: Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.C112.396903

    MARK and its homolog regulate TORC1 activity in Drosophila and mammalian cells. A , Par-1 negatively regulates TORC1 activity in Drosophila S2 cells. D rosophila S2 cells untreated ( NC , lane 1 ) or treated with the double-stranded RNA against individual
    Figure Legend Snippet: MARK and its homolog regulate TORC1 activity in Drosophila and mammalian cells. A , Par-1 negatively regulates TORC1 activity in Drosophila S2 cells. D rosophila S2 cells untreated ( NC , lane 1 ) or treated with the double-stranded RNA against individual

    Techniques Used: Activity Assay

    27) Product Images from "Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export"

    Article Title: Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export

    Journal: Genes & Development

    doi: 10.1101/gad.328690.119

    Nxf3 interacts with Nxt1 at piRNA cluster loci, and piRNA precursor export requires Crm1. ( A ) Expression and localization of Nxf3 and Nxt1 in control and nxf3 mut nurse cell nuclei is shown by immunofluorescence. (Green) Nxf3; (red) Nxt1; (blue) DNA. Scale bar, 2 µm. ( B ) Expression and localization of Nxf3 and Vas in egg chambers upon germline-specific knockdown (GLKD) of nxt1 or w (control) are shown by immunofluorescence. (Green) Nxf3; (magenta) Vas; (blue) DNA. Scale bar, 10 µm. ( C ) Western blot analyses of Flag tag pull-down from lysates of S2 cells transfected with the indicated expression constructs. (IN) Input (10%); (UB) unbound (2.5%); (IP) immunoprecipitate (50%). ( D ) Expression and localization of the Nxf3-GFP-3xFlag construct in S2 cells treated with leptomycin B (LMB) or control treatment are shown by immunofluorescence. (Green) Flag; (red) Lamin; (blue) DNA. Scale bar, 2 µm.
    Figure Legend Snippet: Nxf3 interacts with Nxt1 at piRNA cluster loci, and piRNA precursor export requires Crm1. ( A ) Expression and localization of Nxf3 and Nxt1 in control and nxf3 mut nurse cell nuclei is shown by immunofluorescence. (Green) Nxf3; (red) Nxt1; (blue) DNA. Scale bar, 2 µm. ( B ) Expression and localization of Nxf3 and Vas in egg chambers upon germline-specific knockdown (GLKD) of nxt1 or w (control) are shown by immunofluorescence. (Green) Nxf3; (magenta) Vas; (blue) DNA. Scale bar, 10 µm. ( C ) Western blot analyses of Flag tag pull-down from lysates of S2 cells transfected with the indicated expression constructs. (IN) Input (10%); (UB) unbound (2.5%); (IP) immunoprecipitate (50%). ( D ) Expression and localization of the Nxf3-GFP-3xFlag construct in S2 cells treated with leptomycin B (LMB) or control treatment are shown by immunofluorescence. (Green) Flag; (red) Lamin; (blue) DNA. Scale bar, 2 µm.

    Techniques Used: Expressing, Immunofluorescence, Western Blot, FLAG-tag, Transfection, Construct

    28) Product Images from "Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)"

    Article Title: Analysis of Expression, Cellular Localization, and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072592

    Activation of the promoters of Drosophila and shrimp AMP genes by overexpression of LvIAP2 in Drosophila S2 cells. Drosophila S2 cells were transfected with a protein expression vector (pAC5.1 empty vector or pAC5.1-LvIAP2 vector), a luciferase reporter plasmid (pGL3-Basic, pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs, or pGL3-AttA), and pRL-TK Renilla luciferase plasmid (as an internal control) (Promega, USA). Thirty-six hours later, the cells were harvested for examination of luciferase activities using the dual luciferase reporter assay system (Promega, USA). All data are representative of three independent experiments. The bars indicate the mean ± S.D. of the luciferase activity (n = 3).
    Figure Legend Snippet: Activation of the promoters of Drosophila and shrimp AMP genes by overexpression of LvIAP2 in Drosophila S2 cells. Drosophila S2 cells were transfected with a protein expression vector (pAC5.1 empty vector or pAC5.1-LvIAP2 vector), a luciferase reporter plasmid (pGL3-Basic, pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs, or pGL3-AttA), and pRL-TK Renilla luciferase plasmid (as an internal control) (Promega, USA). Thirty-six hours later, the cells were harvested for examination of luciferase activities using the dual luciferase reporter assay system (Promega, USA). All data are representative of three independent experiments. The bars indicate the mean ± S.D. of the luciferase activity (n = 3).

    Techniques Used: Activation Assay, Over Expression, Transfection, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

    Subcellular localization of LvIAP1 (A), LvIAP2 (B), and LvIAP3 (C) in Drosophila S2 cells. Drosophila S2 cells were transfected with the pAc5.1-LvIAP1-3-GFP plasmids. At 36 hours post-transfection, the cover slips were washed, fixed, and stained with Hoechst 33258. The protein cellular localization was examined under a Leica laser scanning confocal microscope. The nuclei were visualized using the Hoechst stain (blue).
    Figure Legend Snippet: Subcellular localization of LvIAP1 (A), LvIAP2 (B), and LvIAP3 (C) in Drosophila S2 cells. Drosophila S2 cells were transfected with the pAc5.1-LvIAP1-3-GFP plasmids. At 36 hours post-transfection, the cover slips were washed, fixed, and stained with Hoechst 33258. The protein cellular localization was examined under a Leica laser scanning confocal microscope. The nuclei were visualized using the Hoechst stain (blue).

    Techniques Used: Transfection, Staining, Microscopy

    Activation of the promoters of  WSSV069  ( ie1 ),  WSSV303 , and  WSSV371  by overexpression of LvIAP2 in  Drosophila  S2 cells.
    Figure Legend Snippet: Activation of the promoters of WSSV069 ( ie1 ), WSSV303 , and WSSV371 by overexpression of LvIAP2 in Drosophila S2 cells.

    Techniques Used: Activation Assay, Over Expression

    29) Product Images from "Exceptional stability of a perilipin on lipid droplets depends on its polar residues, suggesting multimeric assembly"

    Article Title: Exceptional stability of a perilipin on lipid droplets depends on its polar residues, suggesting multimeric assembly

    Journal: eLife

    doi: 10.7554/eLife.61401

    Cell-to-cell variability in the recovery of Plin4 12mer-GFP after photobleaching of LDs in Drosophila S2 cells. FRAP was performed on oleic acid-induced LDs in S2 cells stably transfected with Plin4 12mer-GFP. ( A ) Fluorescence recovery curves from individual cells in the same experiment performed in control cells (RNAi against luciferase). Each curve represents mean ± SD from FRAP on three LDs in the same cell, as exemplified in the two images below the graph, showing two cells before bleaching. Areas that were bleached are marked with green circles. Fast fluorescence recovery was observed in the cell on the left, slow in the cell on the right. The fluorescence signal in the two images was acquired under the same settings, showing that the total Plin4-GFP expression level in the two cells was similar. However, note the difference in the amount of cytosolic signal. Scale bar: 5 μm. ( B ) Same as A, except that FRAP was performed on cells in which CCT1 has been depleted by RNAi. ( C ) Correlation between the ratio of LD/cytosolic Plin4 12mer-GFP signal and the half-time of recovery, as determined from the curves shown in A and B. Each dot represents one cell, data is from two independent experiments. Asterisks denote cells which had very slow recovery kinetics (half-time > 100 s; in this case the value was set to 100 s).
    Figure Legend Snippet: Cell-to-cell variability in the recovery of Plin4 12mer-GFP after photobleaching of LDs in Drosophila S2 cells. FRAP was performed on oleic acid-induced LDs in S2 cells stably transfected with Plin4 12mer-GFP. ( A ) Fluorescence recovery curves from individual cells in the same experiment performed in control cells (RNAi against luciferase). Each curve represents mean ± SD from FRAP on three LDs in the same cell, as exemplified in the two images below the graph, showing two cells before bleaching. Areas that were bleached are marked with green circles. Fast fluorescence recovery was observed in the cell on the left, slow in the cell on the right. The fluorescence signal in the two images was acquired under the same settings, showing that the total Plin4-GFP expression level in the two cells was similar. However, note the difference in the amount of cytosolic signal. Scale bar: 5 μm. ( B ) Same as A, except that FRAP was performed on cells in which CCT1 has been depleted by RNAi. ( C ) Correlation between the ratio of LD/cytosolic Plin4 12mer-GFP signal and the half-time of recovery, as determined from the curves shown in A and B. Each dot represents one cell, data is from two independent experiments. Asterisks denote cells which had very slow recovery kinetics (half-time > 100 s; in this case the value was set to 100 s).

    Techniques Used: Stable Transfection, Transfection, Fluorescence, Luciferase, Expressing

    30) Product Images from "Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB"

    Article Title: Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB

    Journal: EMBO Reports

    doi: 10.15252/embr.201948335

    GCN5 acetylates TFEB at K116, K274, and K279 (Related to Fig 3 ) Intracellular TFEB level in WT and GCN5 KO HeLa cells, and in HeLa cells transfected with GFP‐GCN5. Quantification of the acetylation levels of TFEB‐Flag and TFEB in Fig 3 E–G. Mass spectrometry analysis of acetylated sites in TFEB. MS/MS spectrum recorded on the [M + 3H] 3+ ion at mass/charge ratio ( m/z ) 3508.7496 of the acetylated peptide ISPAQGSPK(Ac)PPPAASPGVR (E); on the [M + 2H] 2+ ion at m/z 1001.5779 of the acetylated peptide WNK(Ac)GTILK (F); and on the [M + 2H] 2+ ion at m/z 689.3905 of the acetylated peptide GTILK(Ac)ASVDYIR (G). Quantification of the acetylation levels of Flag‐tagged TFEB and TFEB mutants in Fig 3 J. Schematic illustration of the domains within the TFEB protein and alignment of the amino acid sequences of TFEB in human MiT/TFE family members and in other species. AD, activation domain; bHLH, basic helix‐loop‐helix domain; Zip, zipper domain. Yellow indicates the acetylated lysine residues in TFEB identified by mass spectrometry. dMitf acetylation in S2 cells. Flag‐tagged dMitf or dMitf‐2KR was co‐transfected with Myc‐tagged dGcn5 or acetyltransferase‐dead dGcn5‐m in S2 cells after incubation with or without dGcn5 dsRNA for 72 h. dMitf‐Flag was immunoprecipitated from cells using anti‐Flag beads, and the precipitates were analyzed by immunoblotting using anti‐acetyl‐lysine (Ace‐lys). GFP dsRNA were used as a control. dMitf‐2KR: Lys 445 and Lys 450 were replaced by Arg. Knockdown efficiency of dGcn5 dsRNA in (J) examined by RT–qPCR (mean ± SEM; n = 3 independent experiments; *** P
    Figure Legend Snippet: GCN5 acetylates TFEB at K116, K274, and K279 (Related to Fig 3 ) Intracellular TFEB level in WT and GCN5 KO HeLa cells, and in HeLa cells transfected with GFP‐GCN5. Quantification of the acetylation levels of TFEB‐Flag and TFEB in Fig 3 E–G. Mass spectrometry analysis of acetylated sites in TFEB. MS/MS spectrum recorded on the [M + 3H] 3+ ion at mass/charge ratio ( m/z ) 3508.7496 of the acetylated peptide ISPAQGSPK(Ac)PPPAASPGVR (E); on the [M + 2H] 2+ ion at m/z 1001.5779 of the acetylated peptide WNK(Ac)GTILK (F); and on the [M + 2H] 2+ ion at m/z 689.3905 of the acetylated peptide GTILK(Ac)ASVDYIR (G). Quantification of the acetylation levels of Flag‐tagged TFEB and TFEB mutants in Fig 3 J. Schematic illustration of the domains within the TFEB protein and alignment of the amino acid sequences of TFEB in human MiT/TFE family members and in other species. AD, activation domain; bHLH, basic helix‐loop‐helix domain; Zip, zipper domain. Yellow indicates the acetylated lysine residues in TFEB identified by mass spectrometry. dMitf acetylation in S2 cells. Flag‐tagged dMitf or dMitf‐2KR was co‐transfected with Myc‐tagged dGcn5 or acetyltransferase‐dead dGcn5‐m in S2 cells after incubation with or without dGcn5 dsRNA for 72 h. dMitf‐Flag was immunoprecipitated from cells using anti‐Flag beads, and the precipitates were analyzed by immunoblotting using anti‐acetyl‐lysine (Ace‐lys). GFP dsRNA were used as a control. dMitf‐2KR: Lys 445 and Lys 450 were replaced by Arg. Knockdown efficiency of dGcn5 dsRNA in (J) examined by RT–qPCR (mean ± SEM; n = 3 independent experiments; *** P

    Techniques Used: Transfection, Mass Spectrometry, Activation Assay, Incubation, Immunoprecipitation, Quantitative RT-PCR

    31) Product Images from "Regulation of mTORC1 by the Rab and Arf GTPases *"

    Article Title: Regulation of mTORC1 by the Rab and Arf GTPases *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.C110.102483

    Rab and Arf proteins are indispensable in regulating TORC1 activity in Drosophila S2 cells. Drosophila S2 cells untreated ( lane 1 ) or treated with the double-stranded RNA against individual genes (as indicated by the Drosophila genome CG numbers) were
    Figure Legend Snippet: Rab and Arf proteins are indispensable in regulating TORC1 activity in Drosophila S2 cells. Drosophila S2 cells untreated ( lane 1 ) or treated with the double-stranded RNA against individual genes (as indicated by the Drosophila genome CG numbers) were

    Techniques Used: Activity Assay

    32) Product Images from "The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila"

    Article Title: The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.63

    Nprl2 and Nprl3 inhibit TORC1 activity in Drosophila. ( a ) S2 cells were co-transfected with HA-tagged Nprl2 and V5-tagged Nprl3 or lacZ (control) plasmids. Cells were lysed and immunoprecipitated using an anti-V5 antibody. Cell lysates (inputs) and immunoprecipitates (IP) were detected by western blot using anti-HA and anti-V5 antibodies. ( b ) S2 cells were treated with GFP , nprl2 or nprl3 dsRNA for 4 days and then cultured in Schneider's medium plus 10% FBS (AA+) or amino-acid-free Schneider's medium (AA-) for 60 min. The protein levels of p-4E-BP, total 4E-BP and α -Tubulin were determined by western blot. Similar western Blot results were observed in three independent experiments. ( c ) S2 cells were treated with GFP , nprl2 or nprl3 dsRNA for 4 days and then cultured in amino-acid-free Schneider's medium plus 10% FBS for 24 h. Cell size in G1 phase was normalized to GFP dsRNA control. Error bars represent S.D. values from three independent experiments. * P
    Figure Legend Snippet: Nprl2 and Nprl3 inhibit TORC1 activity in Drosophila. ( a ) S2 cells were co-transfected with HA-tagged Nprl2 and V5-tagged Nprl3 or lacZ (control) plasmids. Cells were lysed and immunoprecipitated using an anti-V5 antibody. Cell lysates (inputs) and immunoprecipitates (IP) were detected by western blot using anti-HA and anti-V5 antibodies. ( b ) S2 cells were treated with GFP , nprl2 or nprl3 dsRNA for 4 days and then cultured in Schneider's medium plus 10% FBS (AA+) or amino-acid-free Schneider's medium (AA-) for 60 min. The protein levels of p-4E-BP, total 4E-BP and α -Tubulin were determined by western blot. Similar western Blot results were observed in three independent experiments. ( c ) S2 cells were treated with GFP , nprl2 or nprl3 dsRNA for 4 days and then cultured in amino-acid-free Schneider's medium plus 10% FBS for 24 h. Cell size in G1 phase was normalized to GFP dsRNA control. Error bars represent S.D. values from three independent experiments. * P

    Techniques Used: Activity Assay, Transfection, Immunoprecipitation, Western Blot, Cell Culture

    33) Product Images from "Two Drosophila Ada2 Homologues Function in Different Multiprotein Complexes"

    Article Title: Two Drosophila Ada2 Homologues Function in Different Multiprotein Complexes

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.9.3305-3319.2003

    dAda2b, but not dAda2a, associates with dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1. Affinity-purified antibodies were conjugated to protein A-Sepharose beads and were used in immunoprecipitation assays of 300 μg of nuclear extract from S2 cells. Input, 30 μg of nuclear extract. (A) Western blots immunoprobed for dSpt3 (left) and dTra1 (right). Lane 1, immunoprecipitates from anti-dAda2a (αdAda2a); lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dAda2b. (B) Western blots probed for dAda2a (left) and dAda2b (right). Lane 1, immunoprecipitates from anti-dSpt3; lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dSpt3; lane 7, immunoprecipitates from anti-dGcn5; lane 8, immunoprecipitates from anti-dAda3. hc, IgG heavy chain. (C) Western blots immunolabeled with antibodies against dmTaf5, dmTaf9, and dmTaf10. Lane 1, immunoprecipitates from anti-dAda2b; lane 2, immunoprecipitates from anti-dSpt3; lane 3, immunoprecipitates from anti-dAda3; lane 4, immunoprecipitates from anti-dAda2a.
    Figure Legend Snippet: dAda2b, but not dAda2a, associates with dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1. Affinity-purified antibodies were conjugated to protein A-Sepharose beads and were used in immunoprecipitation assays of 300 μg of nuclear extract from S2 cells. Input, 30 μg of nuclear extract. (A) Western blots immunoprobed for dSpt3 (left) and dTra1 (right). Lane 1, immunoprecipitates from anti-dAda2a (αdAda2a); lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dAda2b. (B) Western blots probed for dAda2a (left) and dAda2b (right). Lane 1, immunoprecipitates from anti-dSpt3; lane 2, immunoprecipitates from anti-dAda2b; lane 3, immunoprecipitates from anti-dGcn5; lane 4, immunoprecipitates from anti-dAda3; lane 5, immunoprecipitates from anti-dAda2a; lane 6, immunoprecipitates from anti-dSpt3; lane 7, immunoprecipitates from anti-dGcn5; lane 8, immunoprecipitates from anti-dAda3. hc, IgG heavy chain. (C) Western blots immunolabeled with antibodies against dmTaf5, dmTaf9, and dmTaf10. Lane 1, immunoprecipitates from anti-dAda2b; lane 2, immunoprecipitates from anti-dSpt3; lane 3, immunoprecipitates from anti-dAda3; lane 4, immunoprecipitates from anti-dAda2a.

    Techniques Used: Affinity Purification, Immunoprecipitation, Western Blot, Immunolabeling

    34) Product Images from "Comparative analysis of tools for live cell imaging of actin network architecture"

    Article Title: Comparative analysis of tools for live cell imaging of actin network architecture

    Journal: Bioarchitecture

    doi: 10.1080/19490992.2014.1047714

    Comparison of live-cell actin probes and phalloidin in Drosophila S2 cells on ConA. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed S2 cells on ConA stably expressing ( A ) eGFP, ( B ) eGFP-actin, ( C ) Utr230-eGFP, ( D ) Utr261-eGFP, ( E ) F-tractin-eGFP and ( F ) Lifeact-eGFP. Scale bars indicate 5 microns.
    Figure Legend Snippet: Comparison of live-cell actin probes and phalloidin in Drosophila S2 cells on ConA. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed S2 cells on ConA stably expressing ( A ) eGFP, ( B ) eGFP-actin, ( C ) Utr230-eGFP, ( D ) Utr261-eGFP, ( E ) F-tractin-eGFP and ( F ) Lifeact-eGFP. Scale bars indicate 5 microns.

    Techniques Used: Stable Transfection, Expressing

    Comparison of live-cell actin probes and phalloidin in Drosophila S2 cells on PDL. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed S2 cells on PDL stably expressing ( A ) eGFP, ( B ) eGFP-actin, ( C ) Utr230-eGFP, ( D ) Utr261-eGFP, ( E ) F-tractin-eGFP and ( F ) Lifeact-eGFP. Scale bars indicate 5 microns.
    Figure Legend Snippet: Comparison of live-cell actin probes and phalloidin in Drosophila S2 cells on PDL. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed S2 cells on PDL stably expressing ( A ) eGFP, ( B ) eGFP-actin, ( C ) Utr230-eGFP, ( D ) Utr261-eGFP, ( E ) F-tractin-eGFP and ( F ) Lifeact-eGFP. Scale bars indicate 5 microns.

    Techniques Used: Stable Transfection, Expressing

    Comparison of mCherry-actin localization with eGFP-tragged actin reporters. mCherry-actin and eGFP reporter localization in fixed S2 cells with corresponding linescans in the lamellum and lamellipod for ( A–B ) F-tractin-eGFP, ( C–D ) Lifeact-eGFP, ( E–F ) Utr261-eGFP, ( G–H ) eGFP actin (control). Scale bars indicate 5 microns.
    Figure Legend Snippet: Comparison of mCherry-actin localization with eGFP-tragged actin reporters. mCherry-actin and eGFP reporter localization in fixed S2 cells with corresponding linescans in the lamellum and lamellipod for ( A–B ) F-tractin-eGFP, ( C–D ) Lifeact-eGFP, ( E–F ) Utr261-eGFP, ( G–H ) eGFP actin (control). Scale bars indicate 5 microns.

    Techniques Used:

    Intensity plot profiles of actin probes and phalloidin in Drosophila S2 cells on ConA Comparison of intensity profiles across lines (shown in yellow) for Alexa 564 phalloidin (top) and eGFP actin reporters (bottom) in fixed S2 cells on ConA. ( A ) eGFP, ( B ) eGFP-actin, ( C ) Utr230-eGFP, ( D ) Utr261-eGFP, ( E ) F-tractin-eGFP and ( F ) Lifeact-eGFP.
    Figure Legend Snippet: Intensity plot profiles of actin probes and phalloidin in Drosophila S2 cells on ConA Comparison of intensity profiles across lines (shown in yellow) for Alexa 564 phalloidin (top) and eGFP actin reporters (bottom) in fixed S2 cells on ConA. ( A ) eGFP, ( B ) eGFP-actin, ( C ) Utr230-eGFP, ( D ) Utr261-eGFP, ( E ) F-tractin-eGFP and ( F ) Lifeact-eGFP.

    Techniques Used:

    35) Product Images from "Rho-kinase Controls Cell Shape Changes during Cytokinesis"

    Article Title: Rho-kinase Controls Cell Shape Changes during Cytokinesis

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2005.12.043

    Other Potential MRLC Kinases Do Not Appear to Contribute Significantly to Furrowing (A) Quantification of the incidence of bi/multinucleate cells induced by rok dsRNA in combination with a control dsRNA against E. coli LacI, or with further additions of Mbs dsRNA, which partially suppresses the rok RNAi phenotype, together with dsRNAs of other putative myosin RLC regulators: bent (Projectin myosin light chain kinase, CG32019); Mlck (stretchin-MLCK, CG18255); Pak (PAK-kinase, p21-activated kinase, CG10296); gek (genghis khan, Myotonic dystrophy kinase related, CG4012); and cit (citron, CG10522). S2 cells were incubated with equal amounts of dsRNA for 4 days, fixed, and stained, and the proportion of multinucleate cells counted. LacI dsRNA was used to standardize the total amounts of RNA added. Mbs RNAi greatly reduced the number of multinucleate cells induced by rok RNAi (compare first two bars). Except for citron, which is itself required for cytokinesis (see below), dsRNAs to other putative MRLC kinases had no effect on the frequency of failed cytokinesis in cells depleted of Rok and Mbs.(B-C) Selected frames from time-lapse sequences of GFP-tubulin S2 cells. (B) A cell following 7 days rok + Mbs RNAi. Cell elongation was perturbed (note the buckled spindle 00:04:39), and furrowing was delayed and slowed, but cytokinesis was successful (see Movie 16). (C) A cell following 4 days rok + Mbs + cit RNAi. As with rok + Mbs RNAi, furrowing was delayed and slowed but nonetheless successful. However, cytokinesis still failed through instability of the intercellular bridge, the phenotype characteristic of citron RNAi alone (see Movie 17).
    Figure Legend Snippet: Other Potential MRLC Kinases Do Not Appear to Contribute Significantly to Furrowing (A) Quantification of the incidence of bi/multinucleate cells induced by rok dsRNA in combination with a control dsRNA against E. coli LacI, or with further additions of Mbs dsRNA, which partially suppresses the rok RNAi phenotype, together with dsRNAs of other putative myosin RLC regulators: bent (Projectin myosin light chain kinase, CG32019); Mlck (stretchin-MLCK, CG18255); Pak (PAK-kinase, p21-activated kinase, CG10296); gek (genghis khan, Myotonic dystrophy kinase related, CG4012); and cit (citron, CG10522). S2 cells were incubated with equal amounts of dsRNA for 4 days, fixed, and stained, and the proportion of multinucleate cells counted. LacI dsRNA was used to standardize the total amounts of RNA added. Mbs RNAi greatly reduced the number of multinucleate cells induced by rok RNAi (compare first two bars). Except for citron, which is itself required for cytokinesis (see below), dsRNAs to other putative MRLC kinases had no effect on the frequency of failed cytokinesis in cells depleted of Rok and Mbs.(B-C) Selected frames from time-lapse sequences of GFP-tubulin S2 cells. (B) A cell following 7 days rok + Mbs RNAi. Cell elongation was perturbed (note the buckled spindle 00:04:39), and furrowing was delayed and slowed, but cytokinesis was successful (see Movie 16). (C) A cell following 4 days rok + Mbs + cit RNAi. As with rok + Mbs RNAi, furrowing was delayed and slowed but nonetheless successful. However, cytokinesis still failed through instability of the intercellular bridge, the phenotype characteristic of citron RNAi alone (see Movie 17).

    Techniques Used: Incubation, Staining

    Anaphase Cell Elongation Requires Both rok and Myosin II Function, but Is Less Dependent on Other Cytokinesis Regulators (A-B) Selected frames from time-lapse sequences of S2 cells expressing Spaghetti squash-GFP (in green and in [Á] and [ B ́ ]) and mRFP-tubulin (in red) and progressing through anaphase. (A) A control cell shows the rapid recruitment of Sqh-GFP to the equatorial cortex (see Movie 8). (B) Eighty-four hour rok RNAi caused a dramatic delay and reduction of cortical Sqh-GFP recruitment (Movie 9). (C) Quantification of Sqh-GFP recruitment to the equatorial cortex in individual control and rok RNAi cells, performed as detailed in the Experimental Procedures. All of the rok RNAi cells ultimately furrowed, as in (B). Data points corresponding to the selected frames in Á and B ́ are indicated ( * , #). (D) Anaphase cell elongation (mean increase in cell length from metaphase ± SE, n = 3-9) was determined from time-lapse sequences of individual cells following the indicated RNAi treatments. (E-I) Selected frames from time-lapse sequences of GFP-tubulin S2 cells. (E) Seventy-two hour spaghetti squash RNAi. The spindle buckled when the poles encountered the cortex, and cell elongation and cytokinesis failed (see Movie 10). (F) Seventy-two hour zipper (zip; myosin heavy chain) RNAi. Similarly, the spindle buckled as it tried to elongate in anaphase; cell elongation and cytokinesis both failed (00:13:52-00:20:48, see Movie 11). (G) Seventy-two hour pebble RNAi. Cell elongation occurred and a furrow initiated but then regressed (see Movie 12). (H) Seventy-two hour RacGAP50C RNAi. Some cell elongation occurred, but furrow ingression did not (see Movie 13). (I) Ninety-six hour diaphanous RNAi. Cell elongation occurred normally; a furrow initiated but then the cortex contracted wildly from side to side for several minutes before subsiding (see Movie 14). Times from the metaphase/anaphase transition are indicated in hr:min:s. Scale bars represent 3 μm.
    Figure Legend Snippet: Anaphase Cell Elongation Requires Both rok and Myosin II Function, but Is Less Dependent on Other Cytokinesis Regulators (A-B) Selected frames from time-lapse sequences of S2 cells expressing Spaghetti squash-GFP (in green and in [Á] and [ B ́ ]) and mRFP-tubulin (in red) and progressing through anaphase. (A) A control cell shows the rapid recruitment of Sqh-GFP to the equatorial cortex (see Movie 8). (B) Eighty-four hour rok RNAi caused a dramatic delay and reduction of cortical Sqh-GFP recruitment (Movie 9). (C) Quantification of Sqh-GFP recruitment to the equatorial cortex in individual control and rok RNAi cells, performed as detailed in the Experimental Procedures. All of the rok RNAi cells ultimately furrowed, as in (B). Data points corresponding to the selected frames in Á and B ́ are indicated ( * , #). (D) Anaphase cell elongation (mean increase in cell length from metaphase ± SE, n = 3-9) was determined from time-lapse sequences of individual cells following the indicated RNAi treatments. (E-I) Selected frames from time-lapse sequences of GFP-tubulin S2 cells. (E) Seventy-two hour spaghetti squash RNAi. The spindle buckled when the poles encountered the cortex, and cell elongation and cytokinesis failed (see Movie 10). (F) Seventy-two hour zipper (zip; myosin heavy chain) RNAi. Similarly, the spindle buckled as it tried to elongate in anaphase; cell elongation and cytokinesis both failed (00:13:52-00:20:48, see Movie 11). (G) Seventy-two hour pebble RNAi. Cell elongation occurred and a furrow initiated but then regressed (see Movie 12). (H) Seventy-two hour RacGAP50C RNAi. Some cell elongation occurred, but furrow ingression did not (see Movie 13). (I) Ninety-six hour diaphanous RNAi. Cell elongation occurred normally; a furrow initiated but then the cortex contracted wildly from side to side for several minutes before subsiding (see Movie 14). Times from the metaphase/anaphase transition are indicated in hr:min:s. Scale bars represent 3 μm.

    Techniques Used: Expressing

    Model for Anaphase Cell Elongation and Initiation of Cytokinesis in Drosophila S2 Cells We propose that both polar relaxation and equatorial contraction contribute to anaphase cell elongation and the initiation of cytokinesis in S2 cells. In our model, the cell undergoes the transition to anaphase with the rigid cortex of a metaphase cell (in black), but this rigidity must be modulated during anaphase. As the chromosomes (in blue) segregate and the anaphase spindle (MTs in green) extends, the polar cortices relax (dotted lines) and a broad contraction begins at the cell equator (in red). Both of these processes, which normally occur around 2-3 min from anaphase onset, require Rok (and Myosin II): As a result, elongation is effectively blocked by depletion of Rok. In contrast, Pebble and RacGAP50C only have an input into the broad equatorial contraction so that depletion does not remove the polar relaxation input into cell elongation. Later, around 5 min after anaphase onset, an actin ring (dotted red line) drives furrow ingression from the center of the earlier broad zone of contractility. Actin ring contraction is sensitive to Pebble, RacGAP50C, and Diaphanous depletion, but Rok depletion, unless severe or prolonged, leads only to a delay in furrowing and modification of the shape of the ingressing furrow. We propose that all three processes of polar relaxation, broad equatorial contraction, and actin ring contraction are promoted by Rok and myosin II, whereas other cytokinesis regulators such as Pebble are more specialized in the promotion of equatorial contraction and actin ring assembly. Accordingly, Rok depletion is particularly effective at blocking/delaying the earliest events in cytokinesis.
    Figure Legend Snippet: Model for Anaphase Cell Elongation and Initiation of Cytokinesis in Drosophila S2 Cells We propose that both polar relaxation and equatorial contraction contribute to anaphase cell elongation and the initiation of cytokinesis in S2 cells. In our model, the cell undergoes the transition to anaphase with the rigid cortex of a metaphase cell (in black), but this rigidity must be modulated during anaphase. As the chromosomes (in blue) segregate and the anaphase spindle (MTs in green) extends, the polar cortices relax (dotted lines) and a broad contraction begins at the cell equator (in red). Both of these processes, which normally occur around 2-3 min from anaphase onset, require Rok (and Myosin II): As a result, elongation is effectively blocked by depletion of Rok. In contrast, Pebble and RacGAP50C only have an input into the broad equatorial contraction so that depletion does not remove the polar relaxation input into cell elongation. Later, around 5 min after anaphase onset, an actin ring (dotted red line) drives furrow ingression from the center of the earlier broad zone of contractility. Actin ring contraction is sensitive to Pebble, RacGAP50C, and Diaphanous depletion, but Rok depletion, unless severe or prolonged, leads only to a delay in furrowing and modification of the shape of the ingressing furrow. We propose that all three processes of polar relaxation, broad equatorial contraction, and actin ring contraction are promoted by Rok and myosin II, whereas other cytokinesis regulators such as Pebble are more specialized in the promotion of equatorial contraction and actin ring assembly. Accordingly, Rok depletion is particularly effective at blocking/delaying the earliest events in cytokinesis.

    Techniques Used: Modification, Blocking Assay

    Rho-kinase Is Required for Anaphase Cell Elongation (A-B) Selected frames from time-lapse sequences of Drosophila S2 cells, stably expressing histone H2B-GFP, progressing through anaphase (time from the metaphase/anaphase transition indicated in hr:min:s). (A) A typical control cell undergoing anaphase cell elongation. Note the appearance of a polar bleb (arrows: 00:01:52, 00:02:08), allowing for the establishment of a new cell boundary to accommodate the segregating DNA masses (00:02:24; see also Movie 1). (B) A cell following 72 hr rok RNAi failed to elongate in anaphase. No polar blebs appeared, and the spindle poles and segregating DNA masses smashed up against the cell cortex, which bulged slightly outward (arrow). Although furrow ingression was delayed and did not initiate within the 6 min of the sequence depicted here, the cell ultimately divided successfully (see Movie 2). (C) Fixed images of S2 cells following 72 hr rok RNAi, stained for DNA (blue), MTs (green), and F-actin (red). Whereas the cell in late anaphase A (left) appears normal, the anaphase B cell (middle) and telophase cell (right) have failed to elongate. Note the buckled appearance of the MTs in the middle panel. (D-F) Selected frames from fluorescent time-lapse sequences of GFP-tubulin expressing S2 cells in anaphase. (D) A typical control cell. As the cell and spindle coordinately elongated in anaphase, polar blebs appeared (arrows), and the centrally localized interpolar MTs elongated parallel to the spindle axis (00:03:30-00:04:54, see Movie 3). (E) A cell following 48 hr rok RNAi. As the separating spindle poles reached the cell cortex, no polar blebs were observed, cell elongation did not keep pace with spindle extension, and the interpolar MTs buckled outwards (arrow, 00:04:08, see Movie 4). (F) A cell following 120 hr rok RNAi. Again, the interpolar MTs buckled outward in the absence of cell elongation (arrow, 00:03:51, see Movie 5). Scale bars represent 3 μm.
    Figure Legend Snippet: Rho-kinase Is Required for Anaphase Cell Elongation (A-B) Selected frames from time-lapse sequences of Drosophila S2 cells, stably expressing histone H2B-GFP, progressing through anaphase (time from the metaphase/anaphase transition indicated in hr:min:s). (A) A typical control cell undergoing anaphase cell elongation. Note the appearance of a polar bleb (arrows: 00:01:52, 00:02:08), allowing for the establishment of a new cell boundary to accommodate the segregating DNA masses (00:02:24; see also Movie 1). (B) A cell following 72 hr rok RNAi failed to elongate in anaphase. No polar blebs appeared, and the spindle poles and segregating DNA masses smashed up against the cell cortex, which bulged slightly outward (arrow). Although furrow ingression was delayed and did not initiate within the 6 min of the sequence depicted here, the cell ultimately divided successfully (see Movie 2). (C) Fixed images of S2 cells following 72 hr rok RNAi, stained for DNA (blue), MTs (green), and F-actin (red). Whereas the cell in late anaphase A (left) appears normal, the anaphase B cell (middle) and telophase cell (right) have failed to elongate. Note the buckled appearance of the MTs in the middle panel. (D-F) Selected frames from fluorescent time-lapse sequences of GFP-tubulin expressing S2 cells in anaphase. (D) A typical control cell. As the cell and spindle coordinately elongated in anaphase, polar blebs appeared (arrows), and the centrally localized interpolar MTs elongated parallel to the spindle axis (00:03:30-00:04:54, see Movie 3). (E) A cell following 48 hr rok RNAi. As the separating spindle poles reached the cell cortex, no polar blebs were observed, cell elongation did not keep pace with spindle extension, and the interpolar MTs buckled outwards (arrow, 00:04:08, see Movie 4). (F) A cell following 120 hr rok RNAi. Again, the interpolar MTs buckled outward in the absence of cell elongation (arrow, 00:03:51, see Movie 5). Scale bars represent 3 μm.

    Techniques Used: Stable Transfection, Expressing, Sequencing, Staining

    36) Product Images from "Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome ▿ 26S Proteasome ▿ †"

    Article Title: Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome ▿ 26S Proteasome ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00799-10

    (A) Soluble  Drosophila  Cnc-C is a proteasome substrate and is stabilized by RNAi depletion of proteasome subunit S5a. A C-terminal V5 epitope tagged Cnc-C construct was transfected into  Drosophila  S2 cells, and cells were divided into different treatment
    Figure Legend Snippet: (A) Soluble Drosophila Cnc-C is a proteasome substrate and is stabilized by RNAi depletion of proteasome subunit S5a. A C-terminal V5 epitope tagged Cnc-C construct was transfected into Drosophila S2 cells, and cells were divided into different treatment

    Techniques Used: Construct, Transfection

    37) Product Images from "A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing"

    Article Title: A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

    Journal: RNA

    doi: 10.1261/rna.1048808

    Investigation of the role of the TAF1 exon 13a 5′ splice site in CPT-induced alternative splicing. ( A ) Shown is base pairing between the exon 13a 5′ splice site (ss) in miniTAF1 pre-mRNAs and U1 snRNAs. Expected base pairs are depicted as vertical lines. Pseudouridines in U1 snRNA are depicted as ψ. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 13a 5′ splice site ( 13a 5′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 13a 5′ splice site. ( E ) qPCR analysis of endogenous TAF1 mRNA levels from cells transfected with wt U1 snRNA plasmid or mut U1 snRNA plasmid. Error bars represent the standard errors of the mean.
    Figure Legend Snippet: Investigation of the role of the TAF1 exon 13a 5′ splice site in CPT-induced alternative splicing. ( A ) Shown is base pairing between the exon 13a 5′ splice site (ss) in miniTAF1 pre-mRNAs and U1 snRNAs. Expected base pairs are depicted as vertical lines. Pseudouridines in U1 snRNA are depicted as ψ. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 13a 5′ splice site ( 13a 5′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 13a 5′ splice site. ( E ) qPCR analysis of endogenous TAF1 mRNA levels from cells transfected with wt U1 snRNA plasmid or mut U1 snRNA plasmid. Error bars represent the standard errors of the mean.

    Techniques Used: Cycling Probe Technology, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay, Real-time Polymerase Chain Reaction

    Investigation of the role of IE-A in CPT-induced-alternative splicing. ( A ) A comparison of wt and mutant IE-A ( IE-A mut ) sequences. Mutated nucleotides are indicated in bold typeface. Lines over codons indicate the tandem repeat sequence with the boldness of the line indicating the degree of match to the most common repeat sequence. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contain RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. In the right panel, lanes 1–6 and 7–12 were from different gels, as indicated by the black bar. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2–4). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the IE-A mutation. Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates mutated IE-A .
    Figure Legend Snippet: Investigation of the role of IE-A in CPT-induced-alternative splicing. ( A ) A comparison of wt and mutant IE-A ( IE-A mut ) sequences. Mutated nucleotides are indicated in bold typeface. Lines over codons indicate the tandem repeat sequence with the boldness of the line indicating the degree of match to the most common repeat sequence. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contain RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. In the right panel, lanes 1–6 and 7–12 were from different gels, as indicated by the black bar. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2–4). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the IE-A mutation. Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates mutated IE-A .

    Techniques Used: Cycling Probe Technology, Mutagenesis, Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay

    Investigation of the role of the TAF1 exon 13a 3′ splice site in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant ( 13a 3′ cons mut ) exon 13a 3′ splice site sequences. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 13a 3′ splice site ( 13a 3′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 13a 3′ splice site.
    Figure Legend Snippet: Investigation of the role of the TAF1 exon 13a 3′ splice site in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant ( 13a 3′ cons mut ) exon 13a 3′ splice site sequences. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 13a 3′ splice site ( 13a 3′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 13a 3′ splice site.

    Techniques Used: Cycling Probe Technology, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay

    MiniTAF1 does not recapitulate IR-induced up-regulation of TAF1-4 splicing. ( A ) qPCR analysis of endogenous TAF1 mRNA levels from mock- or IR-treated cells. Error bars represent the standard errors of the mean. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from mock-treated (mock) S2 cells (lanes 1 , 2 ) or IR-treated S2 cells (lanes 3 , 4 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. Positions of miniTAF1 isoforms are indicated to the right of each panel. ( C ) Quantitation of the effect of IR treatment on miniTAF1 mRNA isoforms ( n = 2).
    Figure Legend Snippet: MiniTAF1 does not recapitulate IR-induced up-regulation of TAF1-4 splicing. ( A ) qPCR analysis of endogenous TAF1 mRNA levels from mock- or IR-treated cells. Error bars represent the standard errors of the mean. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from mock-treated (mock) S2 cells (lanes 1 , 2 ) or IR-treated S2 cells (lanes 3 , 4 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. Positions of miniTAF1 isoforms are indicated to the right of each panel. ( C ) Quantitation of the effect of IR treatment on miniTAF1 mRNA isoforms ( n = 2).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Quantitation Assay

    Investigation of the role of the TAF1 exon 12a 5′ splice site in CPT-induced alternative splicing. ( A ) Shown is base pairing between the exon 12a 5′ splice site (ss) in miniTAF1 pre-mRNAs and U1 snRNA. Expected base pairs are depicted as vertical lines. Pseudouridines in U1 snRNA are depicted as ψ. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 12a 5′ splice site ( 12a 5′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 12a 5′ splice site.
    Figure Legend Snippet: Investigation of the role of the TAF1 exon 12a 5′ splice site in CPT-induced alternative splicing. ( A ) Shown is base pairing between the exon 12a 5′ splice site (ss) in miniTAF1 pre-mRNAs and U1 snRNA. Expected base pairs are depicted as vertical lines. Pseudouridines in U1 snRNA are depicted as ψ. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 12a 5′ splice site ( 12a 5′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 12a 5′ splice site.

    Techniques Used: Cycling Probe Technology, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay

    Investigation of the role of the TAF1 exon 12a 3′ splice site in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant ( 12a 3′ cons mut ) exon 12a 3′ splice site sequences. Arrows indicate the splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 12a 3′ splice site ( 12a 3′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 12a 3′ splice site.
    Figure Legend Snippet: Investigation of the role of the TAF1 exon 12a 3′ splice site in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant ( 12a 3′ cons mut ) exon 12a 3′ splice site sequences. Arrows indicate the splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 12a 3′ splice site ( 12a 3′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 12a 3′ splice site.

    Techniques Used: Cycling Probe Technology, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay

    Investigation of the role of IE-C in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant IE-C ( IE-C mut ) sequences. Mutated nucleotides are indicated in bold typeface. Overlines indicate the tandem repeat sequence. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2–4). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the IE-C mutants. Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated IE-C . ( E ) RT-PCR analysis of abl mRNA isoform expression in CPT-treated S2 cells. Even numbered lanes contained RT-PCR samples in which RT was left out. abl-RB differs from abl-RA ).
    Figure Legend Snippet: Investigation of the role of IE-C in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant IE-C ( IE-C mut ) sequences. Mutated nucleotides are indicated in bold typeface. Overlines indicate the tandem repeat sequence. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2–4). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the IE-C mutants. Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated IE-C . ( E ) RT-PCR analysis of abl mRNA isoform expression in CPT-treated S2 cells. Even numbered lanes contained RT-PCR samples in which RT was left out. abl-RB differs from abl-RA ).

    Techniques Used: Cycling Probe Technology, Mutagenesis, Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay

    Investigation of the role of the TAF1 exon 13 3′ splice site in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant ( 13 3′ cons mut ) exon 13 3′ splice-site sequences. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 13 3′ splice site ( 13 3′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 13 3′ splice site.
    Figure Legend Snippet: Investigation of the role of the TAF1 exon 13 3′ splice site in CPT-induced alternative splicing. ( A ) A comparison of wt and mutant ( 13 3′ cons mut ) exon 13 3′ splice-site sequences. Arrows indicate splice sites and bold typeface indicates mutated nucleotides. ( B ) Analysis of miniTAF1 expression by RT-PCR. On the left is analysis of all four mRNA isoforms, and on the right is analysis of exon 13a-containing mRNA isoforms. Samples were analyzed from untreated (untr) S2 cells (lanes 1 , 2 , 7 , 8 ), DMSO-treated S2 cells (lanes 3 , 4 , 9 , 10 ), or CPT-treated S2 cells (lanes 5 , 6 , 11 , 12 ). Even numbered lanes contained RT-PCR samples in which reverse transcriptase (RT) was left out as a control for plasmid contamination. The transfected plasmid is indicated at the top . Positions of the miniTAF1 mRNA isoforms are shown to the right of each panel. ( C ) Quantitation of miniTAF1 mRNA isoforms from panel B ( n = 2). Error bars represent the standard errors of the mean. ( D ) A summary of the effect of CPT-induced signaling on miniTAF1 alternative splicing in the context of the consensus exon 13 3′ splice site ( 13 3′ cons mut ). Shaded boxes indicate alternative exons 12a and 13a. The open arrowhead indicates the mutated exon 13 3′ splice site.

    Techniques Used: Cycling Probe Technology, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, Quantitation Assay

    38) Product Images from "Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of ?-catenin"

    Article Title: Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of ?-catenin

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

    doi: 10.1073/pnas.0307344101

    Nuclear-cytoplasmic shuttling of Axin. ( A ) 293 cells were transiently transfected with plasmids encoding GFP-Axin or GFP-AxinΔDIX. Thirty-six hours after transfection, cells were mock-treated or treated with 5 ng/ml LMB for 3 h and examined by fluorescence microscopy. ( B ) COS cells were infected with a retrovirus encoding GFP-Axin. Cells were mock-treated or treated with LMB for 3 h and examined by fluorescence microscopy. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole. ( C ) 293 cells were mock-treated or treated with LMB for 3 h and subjected to subcellular fractionation. The levels of endogenous Axin in the nuclear and cytoplasmic fractions were determined by immunoblotting with anti-Axin antibodies. The relative purity of the nuclear and cytoplasmic fractions was confirmed by sequential probing for the nuclear marker lamin B and the cytoplasmic marker α-tubulin. ( D ) Drosophila S2 cells were transiently transfected with a plasmid expressing DAxin-HA. Cells were mock-treated or treated with LMB for 3 h, immunostained with anti-HA monoclonal antibodies and FITC-conjugated secondary antibodies, and examined by confocal microscopy. Cell shapes were examined by phase contrast microscopy.
    Figure Legend Snippet: Nuclear-cytoplasmic shuttling of Axin. ( A ) 293 cells were transiently transfected with plasmids encoding GFP-Axin or GFP-AxinΔDIX. Thirty-six hours after transfection, cells were mock-treated or treated with 5 ng/ml LMB for 3 h and examined by fluorescence microscopy. ( B ) COS cells were infected with a retrovirus encoding GFP-Axin. Cells were mock-treated or treated with LMB for 3 h and examined by fluorescence microscopy. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole. ( C ) 293 cells were mock-treated or treated with LMB for 3 h and subjected to subcellular fractionation. The levels of endogenous Axin in the nuclear and cytoplasmic fractions were determined by immunoblotting with anti-Axin antibodies. The relative purity of the nuclear and cytoplasmic fractions was confirmed by sequential probing for the nuclear marker lamin B and the cytoplasmic marker α-tubulin. ( D ) Drosophila S2 cells were transiently transfected with a plasmid expressing DAxin-HA. Cells were mock-treated or treated with LMB for 3 h, immunostained with anti-HA monoclonal antibodies and FITC-conjugated secondary antibodies, and examined by confocal microscopy. Cell shapes were examined by phase contrast microscopy.

    Techniques Used: Transfection, Fluorescence, Microscopy, Infection, Fractionation, Marker, Plasmid Preparation, Expressing, Confocal Microscopy

    39) Product Images from "Anaphase B spindle dynamics in Drosophila S2 cells: Comparison with embryo spindles"

    Article Title: Anaphase B spindle dynamics in Drosophila S2 cells: Comparison with embryo spindles

    Journal: Cell Division

    doi: 10.1186/1747-1028-6-8

    Fluorescent tubulin speckle trajectories pre-anaphase B and during anaphase B spindle elongation . Kymographs generated from fluorescent speckle time lapse movies of S2 cells expressing low levels of GFP-α-tubulin during pre-anaphase B and anaphase B, illustrating tubulin speckle trajectories (yellow lines).
    Figure Legend Snippet: Fluorescent tubulin speckle trajectories pre-anaphase B and during anaphase B spindle elongation . Kymographs generated from fluorescent speckle time lapse movies of S2 cells expressing low levels of GFP-α-tubulin during pre-anaphase B and anaphase B, illustrating tubulin speckle trajectories (yellow lines).

    Techniques Used: Generated, Expressing

    Timing of anaphase A chromatid-to-pole movement in Drosophila S2 cells . (A) Stills from a movie following anaphase A chromatid-to-pole movement in an S2 cell stably expressing histone 2B (green) and -α-tubulin (red). Time is expressed as min:sec and scale bar represents 5 μm. (B) Graph illustrating the timing of anaphase A chromatid to pole motility relative to anaphase B spindle elongation. Note that time point 0 in the graph does not correspond to that in the stills.
    Figure Legend Snippet: Timing of anaphase A chromatid-to-pole movement in Drosophila S2 cells . (A) Stills from a movie following anaphase A chromatid-to-pole movement in an S2 cell stably expressing histone 2B (green) and -α-tubulin (red). Time is expressed as min:sec and scale bar represents 5 μm. (B) Graph illustrating the timing of anaphase A chromatid to pole motility relative to anaphase B spindle elongation. Note that time point 0 in the graph does not correspond to that in the stills.

    Techniques Used: Stable Transfection, Expressing, Size-exclusion Chromatography

    The suppression of microtubule poleward flux is linked to spindle elongation during anaphase B . Graph illustrating the linear inverse relationship between poleward flux during anaphase B and the rate of anaphase B. Data points display the behaviour of individual fluorescent tubulin speckles within individual spindles of wild-type S2 cells expressing low levels of GFP-α-tubulin. The 95% confidence intervals for the line of best fit are also shown.
    Figure Legend Snippet: The suppression of microtubule poleward flux is linked to spindle elongation during anaphase B . Graph illustrating the linear inverse relationship between poleward flux during anaphase B and the rate of anaphase B. Data points display the behaviour of individual fluorescent tubulin speckles within individual spindles of wild-type S2 cells expressing low levels of GFP-α-tubulin. The 95% confidence intervals for the line of best fit are also shown.

    Techniques Used: Expressing

    Microtubule plus ends redistribute at anaphase B onset . (A and C) Distribution of the microtubule tip tracker EB1 over time in S2 cells stably expressing RFP-EB1. Time is shown in min:sec and scale bars represent 5 μm. Two different phenotypes were observed. Arrow in bottom left panel of C indicates puncta of EB1 fluorescence visible at the equator of individual MT bundles. Tight redistribution of the plus ends (A) to the equator was seen in 25% of cells. (B and D) Whole spindle kymographs demonstrating EB-1 distribution across the spindle over time.
    Figure Legend Snippet: Microtubule plus ends redistribute at anaphase B onset . (A and C) Distribution of the microtubule tip tracker EB1 over time in S2 cells stably expressing RFP-EB1. Time is shown in min:sec and scale bars represent 5 μm. Two different phenotypes were observed. Arrow in bottom left panel of C indicates puncta of EB1 fluorescence visible at the equator of individual MT bundles. Tight redistribution of the plus ends (A) to the equator was seen in 25% of cells. (B and D) Whole spindle kymographs demonstrating EB-1 distribution across the spindle over time.

    Techniques Used: Stable Transfection, Expressing, Size-exclusion Chromatography, Fluorescence

    40) Product Images from "A Phenotype at Last: Essential Role for the Yersinia enterocolitica Ysa Type III Secretion System in a Drosophila melanogaster S2 Cell Model"

    Article Title: A Phenotype at Last: Essential Role for the Yersinia enterocolitica Ysa Type III Secretion System in a Drosophila melanogaster S2 Cell Model

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01454-12

    The Ysa T3SS is necessary for intracellular replication within S2 cells. A gentamicin protection assay was performed using S2 cells seeded on glass coverslips. At the indicated times, samples were washed, fixed, and processed for fluorescence microscopy.
    Figure Legend Snippet: The Ysa T3SS is necessary for intracellular replication within S2 cells. A gentamicin protection assay was performed using S2 cells seeded on glass coverslips. At the indicated times, samples were washed, fixed, and processed for fluorescence microscopy.

    Techniques Used: Fluorescence, Microscopy

    Infection of S2 cells by Y. enterocolitica requires a functional Ysa T3SS. S2 cells were infected with Y. enterocolitica strains at an MOI of 10 and subjected to gentamicin protection assays as described in Materials and Methods. Relative fluorescence
    Figure Legend Snippet: Infection of S2 cells by Y. enterocolitica requires a functional Ysa T3SS. S2 cells were infected with Y. enterocolitica strains at an MOI of 10 and subjected to gentamicin protection assays as described in Materials and Methods. Relative fluorescence

    Techniques Used: Infection, Functional Assay, Fluorescence

    Model of Ysa-dependent  Y. enterocolitica  infection of S2 cells. The three phases of infection are indicated, showing the influence of a subset of Ysps on internalization (1), replication and presumptive escape from intracellular compartment (2), followed
    Figure Legend Snippet: Model of Ysa-dependent Y. enterocolitica infection of S2 cells. The three phases of infection are indicated, showing the influence of a subset of Ysps on internalization (1), replication and presumptive escape from intracellular compartment (2), followed

    Techniques Used: Infection

    Loss of the known Ysa effector proteins does not impair bacterial replication within S2 cells. A gentamicin protection assay was performed as described for  using pYV −  strains carrying in-frame deletions of an individual  ysp  gene (Δ
    Figure Legend Snippet: Loss of the known Ysa effector proteins does not impair bacterial replication within S2 cells. A gentamicin protection assay was performed as described for using pYV − strains carrying in-frame deletions of an individual ysp gene (Δ

    Techniques Used:

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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
    https://www.bioz.com/result/blasticidin s hcl/product/Thermo Fisher
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    blasticidin s hcl - by Bioz Stars, 2022-01
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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
    Dcv Infected Drosophila S2 Cells, 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
    https://www.bioz.com/result/dcv infected drosophila s2 cells/product/Thermo Fisher
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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

    Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .

    Journal: Immunity

    Article Title: Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

    doi: 10.1016/j.immuni.2017.09.011

    Figure Lengend Snippet: Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .

    Article Snippet: Drosophila S2* cells were cultured in Schneider’s Drosophila medium (GIBCO) supplemented with 10% FBS (not heat-inactivated), 1% Glutamax (GIBCO), and 0.2% PenStrep (GIBCO) at 27°C.

    Techniques: Inhibition, Radial Immuno Diffusion, Activation Assay, Recombinant, Quantitative RT-PCR, Expressing, Injection, Real-time Polymerase Chain Reaction

    PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p

    Journal: Immunity

    Article Title: Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

    doi: 10.1016/j.immuni.2017.09.011

    Figure Lengend Snippet: PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p

    Article Snippet: Drosophila S2* cells were cultured in Schneider’s Drosophila medium (GIBCO) supplemented with 10% FBS (not heat-inactivated), 1% Glutamax (GIBCO), and 0.2% PenStrep (GIBCO) at 27°C.

    Techniques: Radial Immuno Diffusion, Expressing, Mutagenesis, Positive Control, Transfection, Fluorescence