gfp  (Roche)


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    Structured Review

    Roche gfp
    Colocalization and effects of direct NPFFR2 signalling on <t>NPY</t> neurons. a Representative image of <t>GFP</t> expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p
    Gfp, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Diet-induced adaptive thermogenesis requires neuropeptide FF receptor-2 signalling"

    Article Title: Diet-induced adaptive thermogenesis requires neuropeptide FF receptor-2 signalling

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06462-0

    Colocalization and effects of direct NPFFR2 signalling on NPY neurons. a Representative image of GFP expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p
    Figure Legend Snippet: Colocalization and effects of direct NPFFR2 signalling on NPY neurons. a Representative image of GFP expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p

    Techniques Used: Expressing, Immunoprecipitation, Isolation, Mouse Assay

    2) Product Images from "The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †"

    Article Title: The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †

    Journal:

    doi: 10.1128/MCB.25.11.4792-4803.2005

    The MAPK-binding site on MKP-1 contributes to nuclear accumulation. (A) Schematic representation of the basic cluster (RRR) on MKP-1 that resides between the CH2A and CH2B domains. This RRR basic cluster was mutated to ASA in the context of GFP-MKP-1
    Figure Legend Snippet: The MAPK-binding site on MKP-1 contributes to nuclear accumulation. (A) Schematic representation of the basic cluster (RRR) on MKP-1 that resides between the CH2A and CH2B domains. This RRR basic cluster was mutated to ASA in the context of GFP-MKP-1

    Techniques Used: Binding Assay

    The NH 2 terminus of MKP-1 is required for nuclear localization. (A to D) COS-7 cells were transiently transfected with GFP-MKP-1 (A), GFP- MKP-1 47-367 (B), GFP-MKP-1 Δ47-136 (C), or GFP-MKP-1 137-367 (D). Confocal imaging was used to visualize GFP
    Figure Legend Snippet: The NH 2 terminus of MKP-1 is required for nuclear localization. (A to D) COS-7 cells were transiently transfected with GFP-MKP-1 (A), GFP- MKP-1 47-367 (B), GFP-MKP-1 Δ47-136 (C), or GFP-MKP-1 137-367 (D). Confocal imaging was used to visualize GFP

    Techniques Used: Transfection, Imaging

    Generation of GFP-MKP-1 fusion proteins. (A) Schematic representation of the GFP fusion proteins of MKP-1 used in this study. (B) COS-7 cells were either left untransfected or were transfected with expression vectors encoding the GFP-MKP-1 fusion proteins
    Figure Legend Snippet: Generation of GFP-MKP-1 fusion proteins. (A) Schematic representation of the GFP fusion proteins of MKP-1 used in this study. (B) COS-7 cells were either left untransfected or were transfected with expression vectors encoding the GFP-MKP-1 fusion proteins

    Techniques Used: Transfection, Expressing

    The NH 2 terminus of MKP-1 is sufficient for nuclear targeting. GFP-MKP-1 1-46 (A), GFP-MKP-1 47-136 (B), and GFP-MKP-1 1-136 (C) were transiently transfected into COS-7 cells, and confocal imaging was performed to visualize for GFP. The graphs below each
    Figure Legend Snippet: The NH 2 terminus of MKP-1 is sufficient for nuclear targeting. GFP-MKP-1 1-46 (A), GFP-MKP-1 47-136 (B), and GFP-MKP-1 1-136 (C) were transiently transfected into COS-7 cells, and confocal imaging was performed to visualize for GFP. The graphs below each

    Techniques Used: Transfection, Imaging

    The NH 2 terminus of MKP-1 inhibits SRE-mediated activation by preventing Elk-1 phosphorylation. (A) Serum-deprived 293 cells expressing the indicated GFP-MKP-1 fusion proteins along with Flag-Elk-1 were stimulated with 10% FBS for 30 min. Whole-cell lysates
    Figure Legend Snippet: The NH 2 terminus of MKP-1 inhibits SRE-mediated activation by preventing Elk-1 phosphorylation. (A) Serum-deprived 293 cells expressing the indicated GFP-MKP-1 fusion proteins along with Flag-Elk-1 were stimulated with 10% FBS for 30 min. Whole-cell lysates

    Techniques Used: Activation Assay, Expressing

    3) Product Images from "An oscillating Min system in Bacillus subtilis influences asymmetrical septation during sporulation"

    Article Title: An oscillating Min system in Bacillus subtilis influences asymmetrical septation during sporulation

    Journal: Microbiology

    doi: 10.1099/mic.0.059295-0

    E. coli MinD can oscillate in the presence of MinE in B. subtilis. (a) Fluorescence micrographs showing localization of YFP–MinD Ec in B. subtilis strain IB1242 (Δ minD Bs Δ divIVA yfp–minD Ec minE ). In most cells, oscillation of YFP fluorescence could be observed, although in some cells the fluorescence signal appears in the form of dots with reduced mobility. The images were taken with an Olympus BX61 microscope. Two pictures were taken 1.5 min apart. Scale bar, 5 µm. (b) Localization of YFP–MinD Ec in a single cell of strain IB1230 (Δ minD Bs yfp–minD Ec minE ). Images were captured using an Olympus BX61 microscope over a period of 9 min and the frames were deconvolved using Huygens Essential software. Scale bar, 1 µm. (c) Relative quantification of YFP–MinD (upper band) and MinE–GFP (lower band, lanes 1–3) in B. subtilis and MinE–CFP (lower band, lane 6) in E. coli by Western blotting. Anti-GFP antibody was used for detection of YFP–MinD, MinE–GFP and MinE–CFP. Lanes 1–3 represent B. subtilis strain IB1155 (Δ minD Bs yfp–minD Ec minE–gfp ) in which expression of yfp–minD is induced with 0.5 mM IPTG and minE–gfp is induced with three different concentrations of xylose, ranging from 0.05 to 0.3 %. Lane 4 represents a negative control, strain IB1056 (Δ minD Bs ). Lane 5 is strain IB1230 (Δ minD Bs yfp–minD Ec minE ) with expression induced using 0.5 mM IPTG and 0.1 % xylose. Lane 6 represents E. coli strain YLS1 : : pYLS68 grown as described elsewhere ( Shih et al. , 2002 ).
    Figure Legend Snippet: E. coli MinD can oscillate in the presence of MinE in B. subtilis. (a) Fluorescence micrographs showing localization of YFP–MinD Ec in B. subtilis strain IB1242 (Δ minD Bs Δ divIVA yfp–minD Ec minE ). In most cells, oscillation of YFP fluorescence could be observed, although in some cells the fluorescence signal appears in the form of dots with reduced mobility. The images were taken with an Olympus BX61 microscope. Two pictures were taken 1.5 min apart. Scale bar, 5 µm. (b) Localization of YFP–MinD Ec in a single cell of strain IB1230 (Δ minD Bs yfp–minD Ec minE ). Images were captured using an Olympus BX61 microscope over a period of 9 min and the frames were deconvolved using Huygens Essential software. Scale bar, 1 µm. (c) Relative quantification of YFP–MinD (upper band) and MinE–GFP (lower band, lanes 1–3) in B. subtilis and MinE–CFP (lower band, lane 6) in E. coli by Western blotting. Anti-GFP antibody was used for detection of YFP–MinD, MinE–GFP and MinE–CFP. Lanes 1–3 represent B. subtilis strain IB1155 (Δ minD Bs yfp–minD Ec minE–gfp ) in which expression of yfp–minD is induced with 0.5 mM IPTG and minE–gfp is induced with three different concentrations of xylose, ranging from 0.05 to 0.3 %. Lane 4 represents a negative control, strain IB1056 (Δ minD Bs ). Lane 5 is strain IB1230 (Δ minD Bs yfp–minD Ec minE ) with expression induced using 0.5 mM IPTG and 0.1 % xylose. Lane 6 represents E. coli strain YLS1 : : pYLS68 grown as described elsewhere ( Shih et al. , 2002 ).

    Techniques Used: Fluorescence, Microscopy, Software, Western Blot, Expressing, Negative Control

    4) Product Images from "Recruitment and Activation of RSK2 by HIV-1 Tat"

    Article Title: Recruitment and Activation of RSK2 by HIV-1 Tat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000151

    Suppression of HIV transcription by FMK, a small-molecule inhibitor of RSK2. (A) Transient transfection of Jurkat 1G5 cells, containing an integrated HIV LTR luciferase construct, with Tat/FLAG (20 and 200 ng). Transfected cells were treated with indicated amounts of FMK or DMSO for 18 h. Values are mean±SEM of four experiments. (B) Cotransfection of Jurkat T cells with 5xUAS luciferase and Gal4-CDK9 (20 ng) and subsequent treatment with FMK at indicated concentrations. Values are means±SEM of four experiments. (C) GFP expression in Jurkat T cells infected with HIV NL4-3 containing the GFP open reading frame in place of the viral nef gene or with an HIV-based lentiviral vector expressing GFP from the heterologous EF-1α promoter. After overnight infection, cells were treated with FMK or DMSO for 36 h. Values are means±SEM of three experiments. *p = 0.002 ( t test). (D) GFP expression in one representative experiment performed with HIV GFP or HIV (EF-1α) GFP virus.
    Figure Legend Snippet: Suppression of HIV transcription by FMK, a small-molecule inhibitor of RSK2. (A) Transient transfection of Jurkat 1G5 cells, containing an integrated HIV LTR luciferase construct, with Tat/FLAG (20 and 200 ng). Transfected cells were treated with indicated amounts of FMK or DMSO for 18 h. Values are mean±SEM of four experiments. (B) Cotransfection of Jurkat T cells with 5xUAS luciferase and Gal4-CDK9 (20 ng) and subsequent treatment with FMK at indicated concentrations. Values are means±SEM of four experiments. (C) GFP expression in Jurkat T cells infected with HIV NL4-3 containing the GFP open reading frame in place of the viral nef gene or with an HIV-based lentiviral vector expressing GFP from the heterologous EF-1α promoter. After overnight infection, cells were treated with FMK or DMSO for 36 h. Values are means±SEM of three experiments. *p = 0.002 ( t test). (D) GFP expression in one representative experiment performed with HIV GFP or HIV (EF-1α) GFP virus.

    Techniques Used: Transfection, Luciferase, Construct, Cotransfection, Expressing, Infection, Plasmid Preparation

    5) Product Images from "Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus"

    Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54766-y

    Gdf11 -IRES-GFP expression is primarily detected within T and B lymphocytes of the peripheral blood. ( A ) Representative flow cytometry analysis of GFP expression within CD3 + T cells, CD19 + B cells, CD11b + /Ly6G − monocytes and CD11b + /Ly6G + neutrophils from peripheral blood. ( B ) Quantification of GFP+ T cells, B cells, monocytes and neutrophils in 2 month old mice from lines 1B, 11 and 12 and WT controls. N = 3–8 males and 3–8 females per genotype. Circles: males. Triangles: Females. Individual data points overlaid with mean ± SD. ( C ) Real time PCR analysis of Gdf11 levels in CD19+ and CD19- splenic cells from young (2-month old) and aged (24-month old) mice. Hprt was used as a housekeeping gene. ( D,E ) Quantification of ( D ), GFP + peripheral blood T cells and ( E ), GFP + peripheral blood B cells within heterozygous mice from line 1B during aging. ( F,G ) Quantification of GFP mean fluorescence intensity within F , peripheral blood T cells and ( G ), peripheral blood B cells in heterozygous mice from line 1B during aging. Mean fluorescence intensity (MFI) values normalized to wild type mice for each timepoint. ( H ) Quantification of total T cell frequency (red) and B cell frequency (blue) out of live peripheral blood cells during aging. N = 25 males and 19 females. Data points represent mean with error bars denoting SEM.
    Figure Legend Snippet: Gdf11 -IRES-GFP expression is primarily detected within T and B lymphocytes of the peripheral blood. ( A ) Representative flow cytometry analysis of GFP expression within CD3 + T cells, CD19 + B cells, CD11b + /Ly6G − monocytes and CD11b + /Ly6G + neutrophils from peripheral blood. ( B ) Quantification of GFP+ T cells, B cells, monocytes and neutrophils in 2 month old mice from lines 1B, 11 and 12 and WT controls. N = 3–8 males and 3–8 females per genotype. Circles: males. Triangles: Females. Individual data points overlaid with mean ± SD. ( C ) Real time PCR analysis of Gdf11 levels in CD19+ and CD19- splenic cells from young (2-month old) and aged (24-month old) mice. Hprt was used as a housekeeping gene. ( D,E ) Quantification of ( D ), GFP + peripheral blood T cells and ( E ), GFP + peripheral blood B cells within heterozygous mice from line 1B during aging. ( F,G ) Quantification of GFP mean fluorescence intensity within F , peripheral blood T cells and ( G ), peripheral blood B cells in heterozygous mice from line 1B during aging. Mean fluorescence intensity (MFI) values normalized to wild type mice for each timepoint. ( H ) Quantification of total T cell frequency (red) and B cell frequency (blue) out of live peripheral blood cells during aging. N = 25 males and 19 females. Data points represent mean with error bars denoting SEM.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Real-time Polymerase Chain Reaction, Fluorescence

    Generation of Gdf11 -IRES-GFP knock-in reporter mice using CRISPR/Cas9. ( A) Schematic of Gdf11 -IRES-GFP targeting to the Gdf11 locus. Blue underlined text indicates the protospacer adjacent motif (PAM) for sgRNA3. Red text indicates the target sequence for sgRNA3. Black arrowhead indicates the predicted cut site for sgRNA3. Primers used for PCR-based screening are designated as A, B, C, D, and E above each allele, and predicted amplicon sizes are listed beneath each allele. The location of NcoI restriction sites and Southern blot probe sequences are indicated in red and blue text, respectively. HA-L: Left homology arm. HA-R: Right homology arm. ( B ) PCR screening of 5 founder mice from Round #1 of injections using primer pair A–C. Expected size: WT = 3.1 kb; KI = 4.3 kb. Gel image is uncropped with the entirety of the captured image shown. ( C) Chromatogram illustrating sequence of boundaries between top: left homology arm (HA-L) and IRES-GFP, and bottom: IRES-GFP and right homology arm (HA-R). ( D) PCR screening of 36 founder mice from Rounds #3 and #4 of using primer pair B-C. Expected size: WT = 0.8 kb; KI = 2.1 kb; *Non-specific band. Green boxes indicate founder animals harboring the Gdf11 -IRES-GFP knock-in allele. Red boxes indicate founder animals harboring large deletions in Gdf11 . Gel image is uncropped. Positive and negative control reactions for PCR amplification were run on a separate gel, which is presented in Supplementary Fig. 12A .
    Figure Legend Snippet: Generation of Gdf11 -IRES-GFP knock-in reporter mice using CRISPR/Cas9. ( A) Schematic of Gdf11 -IRES-GFP targeting to the Gdf11 locus. Blue underlined text indicates the protospacer adjacent motif (PAM) for sgRNA3. Red text indicates the target sequence for sgRNA3. Black arrowhead indicates the predicted cut site for sgRNA3. Primers used for PCR-based screening are designated as A, B, C, D, and E above each allele, and predicted amplicon sizes are listed beneath each allele. The location of NcoI restriction sites and Southern blot probe sequences are indicated in red and blue text, respectively. HA-L: Left homology arm. HA-R: Right homology arm. ( B ) PCR screening of 5 founder mice from Round #1 of injections using primer pair A–C. Expected size: WT = 3.1 kb; KI = 4.3 kb. Gel image is uncropped with the entirety of the captured image shown. ( C) Chromatogram illustrating sequence of boundaries between top: left homology arm (HA-L) and IRES-GFP, and bottom: IRES-GFP and right homology arm (HA-R). ( D) PCR screening of 36 founder mice from Rounds #3 and #4 of using primer pair B-C. Expected size: WT = 0.8 kb; KI = 2.1 kb; *Non-specific band. Green boxes indicate founder animals harboring the Gdf11 -IRES-GFP knock-in allele. Red boxes indicate founder animals harboring large deletions in Gdf11 . Gel image is uncropped. Positive and negative control reactions for PCR amplification were run on a separate gel, which is presented in Supplementary Fig. 12A .

    Techniques Used: Knock-In, Mouse Assay, CRISPR, Sequencing, Polymerase Chain Reaction, Amplification, Southern Blot, Negative Control

    Validation of Gdf11 -IRES-GFP knock-in reporter mouse lines. ( A , B ) Southern blot analysis of ( A ), Gdf11- IRES-GFP targeted founder mice and ( B ), Gdf11- IRES-GFP F1 progeny. Nco1-digested genomic DNA was hybridized with the internal probe. Expected fragment size: WT = n/a; T (targeted) = 3.5 kb. AI: Additional integration. Blot images were cropped to focus on the target bands. Uncropped blots are presented in Supplementary Fig. 12B,C . ( C ) TLA sequencing coverage and analysis plots from line 1B using outward facing primers residing in the GFP transgene. ( D ) Flow cytometry analysis of GFP expression in live (7AAD − ) peripheral blood cells in left : mice exhibiting correct targeting (lines 1B, 11 and 12) and right: mice exhibiting incorrect targeting (lines 1A and 13). ( E,F ) Real time PCR analysis of Gdf11 levels in FACS-purified GFP high and GFP low splenocytes from line 1B using ( E ), primers spanning exons 1-2 and ( F ), primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in GFP low splenocytes. N = 4 males (blue), 4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( G,H ) Real time PCR analysis of Gdf11 levels in whole spleen from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. Relative Gdf11 expression levels were assayed using ( G ), primers spanning exons 1–2 and ( H) , primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in C57BL/6J mice. N = 3–4 males (blue), 3–4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( I,J ), Quantification of ( I ), GDF11 protein levels, and ( J) , GDF8 protein levels, in serum from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. ( K,L ) Whole mount in situ hybridization for Gdf11 (top) and Gfp (bottom) in E10.5 Gdf11 +/+ and Gdf11 KI/+ embryos from line 1B. For each embryo, the right-most images show the dissected forelimb. mb: midbrain, fb: forebrain, psm: pre-somitic mesoderm, fl: forelimb, hl: hindlimb, s: somite. Scale bar: 0.5 mm.
    Figure Legend Snippet: Validation of Gdf11 -IRES-GFP knock-in reporter mouse lines. ( A , B ) Southern blot analysis of ( A ), Gdf11- IRES-GFP targeted founder mice and ( B ), Gdf11- IRES-GFP F1 progeny. Nco1-digested genomic DNA was hybridized with the internal probe. Expected fragment size: WT = n/a; T (targeted) = 3.5 kb. AI: Additional integration. Blot images were cropped to focus on the target bands. Uncropped blots are presented in Supplementary Fig. 12B,C . ( C ) TLA sequencing coverage and analysis plots from line 1B using outward facing primers residing in the GFP transgene. ( D ) Flow cytometry analysis of GFP expression in live (7AAD − ) peripheral blood cells in left : mice exhibiting correct targeting (lines 1B, 11 and 12) and right: mice exhibiting incorrect targeting (lines 1A and 13). ( E,F ) Real time PCR analysis of Gdf11 levels in FACS-purified GFP high and GFP low splenocytes from line 1B using ( E ), primers spanning exons 1-2 and ( F ), primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in GFP low splenocytes. N = 4 males (blue), 4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( G,H ) Real time PCR analysis of Gdf11 levels in whole spleen from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. Relative Gdf11 expression levels were assayed using ( G ), primers spanning exons 1–2 and ( H) , primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in C57BL/6J mice. N = 3–4 males (blue), 3–4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( I,J ), Quantification of ( I ), GDF11 protein levels, and ( J) , GDF8 protein levels, in serum from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. ( K,L ) Whole mount in situ hybridization for Gdf11 (top) and Gfp (bottom) in E10.5 Gdf11 +/+ and Gdf11 KI/+ embryos from line 1B. For each embryo, the right-most images show the dissected forelimb. mb: midbrain, fb: forebrain, psm: pre-somitic mesoderm, fl: forelimb, hl: hindlimb, s: somite. Scale bar: 0.5 mm.

    Techniques Used: Knock-In, Southern Blot, Mouse Assay, Sequencing, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, FACS, Purification, In Situ Hybridization

    6) Product Images from "Iron Supply via NCOA4-Mediated Ferritin Degradation Maintains Mitochondrial Functions"

    Article Title: Iron Supply via NCOA4-Mediated Ferritin Degradation Maintains Mitochondrial Functions

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00010-19

    The ferritin light chain is required for iron supply to mitochondria under iron-deprived conditions. (A) Wild-type (WT) and FTL knockout (KO) HeLa cells treated with 100 μM DFO for the indicated times were immunoblotted with the indicated antibodies. (B) The PNS was obtained from WT and FTL KO HeLa cells treated with 100 μM DFO for 24 h. Shown are results from BN-PAGE followed by immunoblotting with the indicated antibodies. (C) Representative images of WT and FTL KO cells incubated with 100 μM DFO for 24 h and immunostained with anti-LAMP2 and anti-FTH1 antibodies. Bar = 10 μm. (D) WT and FTL KO HeLa cells expressing GFP-NCOA4 incubated with 100 μM DFO for 24 h. Immunostaining with anti-FTH1 antibodies was performed. Arrows indicate GFP-NCOA4 puncta that colocalized with FTH1. Bars = 10 μm. (E) FTL WT and KO cells expressing GFP-NCOA4 or GFP were incubated for 48 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-GFP magnetic beads. Immunoprecipitates were analyzed by Western blotting. (F) OCRs in WT and FTL KO cells were measured. OCRs were obtained from four independent wells. (G) Results of quantification of basal respiration, ATP production, and maximum respiration. The results are from five independent wells. (H) Mitochondria were isolated from FTL WT and KO cells expressing FLAG-FTL or an empty vector incubated with 100 μM DFO for 6 h. BN-PAGE and SDS-PAGE followed by immunoblotting were performed with the indicated antibodies. (I) FTL KO cells, FLAG-FTL cells, or empty vector cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (J and K) FTL KO cells were transfected with FLAG-FTL or an empty vector with (J) or without (K) GFP-NCOA4. Cells treated with 100 μM DFO for 24 h were immunostained with anti-LAMP2 and anti-FTH1 (K) or anti-FTH1 (J) antibodies. Bars = 10 μm. (L) OCRs in FTL KO cells transfected with FLAG-FTL or an empty vector were measured. OCRs were obtained from five independent wells. (M) Results of quantification of basal respiration, ATP production, and maximum respiration from panel L. Error bars represent mean values ± SEM. ns, not significant; *, P
    Figure Legend Snippet: The ferritin light chain is required for iron supply to mitochondria under iron-deprived conditions. (A) Wild-type (WT) and FTL knockout (KO) HeLa cells treated with 100 μM DFO for the indicated times were immunoblotted with the indicated antibodies. (B) The PNS was obtained from WT and FTL KO HeLa cells treated with 100 μM DFO for 24 h. Shown are results from BN-PAGE followed by immunoblotting with the indicated antibodies. (C) Representative images of WT and FTL KO cells incubated with 100 μM DFO for 24 h and immunostained with anti-LAMP2 and anti-FTH1 antibodies. Bar = 10 μm. (D) WT and FTL KO HeLa cells expressing GFP-NCOA4 incubated with 100 μM DFO for 24 h. Immunostaining with anti-FTH1 antibodies was performed. Arrows indicate GFP-NCOA4 puncta that colocalized with FTH1. Bars = 10 μm. (E) FTL WT and KO cells expressing GFP-NCOA4 or GFP were incubated for 48 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-GFP magnetic beads. Immunoprecipitates were analyzed by Western blotting. (F) OCRs in WT and FTL KO cells were measured. OCRs were obtained from four independent wells. (G) Results of quantification of basal respiration, ATP production, and maximum respiration. The results are from five independent wells. (H) Mitochondria were isolated from FTL WT and KO cells expressing FLAG-FTL or an empty vector incubated with 100 μM DFO for 6 h. BN-PAGE and SDS-PAGE followed by immunoblotting were performed with the indicated antibodies. (I) FTL KO cells, FLAG-FTL cells, or empty vector cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (J and K) FTL KO cells were transfected with FLAG-FTL or an empty vector with (J) or without (K) GFP-NCOA4. Cells treated with 100 μM DFO for 24 h were immunostained with anti-LAMP2 and anti-FTH1 (K) or anti-FTH1 (J) antibodies. Bars = 10 μm. (L) OCRs in FTL KO cells transfected with FLAG-FTL or an empty vector were measured. OCRs were obtained from five independent wells. (M) Results of quantification of basal respiration, ATP production, and maximum respiration from panel L. Error bars represent mean values ± SEM. ns, not significant; *, P

    Techniques Used: Knock-Out, Polyacrylamide Gel Electrophoresis, Incubation, Expressing, Immunostaining, Immunoprecipitation, Magnetic Beads, Western Blot, Isolation, Plasmid Preparation, SDS Page, Transfection

    7) Product Images from "The P25 Protein of Potato Virus X (PVX) Is the Main Pathogenicity Determinant Responsible for Systemic Necrosis in PVX-Associated Synergisms"

    Article Title: The P25 Protein of Potato Virus X (PVX) Is the Main Pathogenicity Determinant Responsible for Systemic Necrosis in PVX-Associated Synergisms

    Journal: Journal of Virology

    doi: 10.1128/JVI.02896-14

    Expression of PVX P25 by a PPV vector increases viral symptoms in Nicotiana benthamiana . (A) Representative disease symptoms induced in N. benthamiana plants by infection with PPV-P25 or PPV-GFP recombinant viruses at 15 days postagroinoculation (dpa). (B) Detached, upper leaves of PPV-P25- and PPV-GFP-infected plants were stained with DAB solution at 15 dpa (upper panels). Representative phenotypes were photographed with a Leica L2 stereoscope (lower panels). Scale bars, 0.5 mm. (C) Western blot analysis of extracts derived from upper leaves of plants infected with PPV-P25 or PPV-GFP at 15 dpa, using antibodies against the HA epitope (left panel) or PPV CP (right panel). The lower panels show the Ponceau S-stained membrane after blotting, as controls of loading.
    Figure Legend Snippet: Expression of PVX P25 by a PPV vector increases viral symptoms in Nicotiana benthamiana . (A) Representative disease symptoms induced in N. benthamiana plants by infection with PPV-P25 or PPV-GFP recombinant viruses at 15 days postagroinoculation (dpa). (B) Detached, upper leaves of PPV-P25- and PPV-GFP-infected plants were stained with DAB solution at 15 dpa (upper panels). Representative phenotypes were photographed with a Leica L2 stereoscope (lower panels). Scale bars, 0.5 mm. (C) Western blot analysis of extracts derived from upper leaves of plants infected with PPV-P25 or PPV-GFP at 15 dpa, using antibodies against the HA epitope (left panel) or PPV CP (right panel). The lower panels show the Ponceau S-stained membrane after blotting, as controls of loading.

    Techniques Used: Expressing, Plasmid Preparation, Infection, Recombinant, Staining, Western Blot, Derivative Assay

    The HR-like response correlates with the silencing suppression activity of P25. (A) Leaves of N. benthamiana were infiltrated with GUS alone or combinations of Agrobacterium cultures containing binary constructs expressing a free GFP reporter gene plus T7-tagged versions of either P25wt or P25 A104V, T117A, K124E mutants or P25stop mutant, as indicated. Photographs were taken with long-wavelength UV light at 3 days postagroinoculation (dpa) (upper panels). Total RNA (10 μg) extracted from infiltrated tissues was hybridized with a probe complementary to GFP (lower panel). Ethidium bromide staining of 25S rRNA is shown as a loading control. (B) Western blot analysis of extracts derived from leaf patches infiltrated with combinations of PPV HCwt plus either P25 wt, A104V, T117A, K124E, P25stop, or GUS at 6 dpa, using antibodies against the T7 epitope. The lower panel shows the Ponceau S-stained membrane after blotting, as a loading control. (C) Leaves were infiltrated with GUS alone or combinations of Agrobacterium cultures containing PPV HCwt plus either T7-tagged versions of P25 wt, A104V, T117A, K124E, P25stop, or GUS. Leaves were stained with DAB solution at 9 dpa. (D) Electrolyte leakage from leaf disks infiltrated with T7-tagged versions of P25 wt, A104V, T117A, K124E, or P25stop, either alone or in combination with PPV HCwt at 9 dpa. GUS alone was infiltrated as a control. Data represent the means ± standard errors for 18 replicates, each consisting of four plants that received the same treatment in three independent experiments. Statistically significant differences between means were determined by employing Scheffé's multiple-range test. Different letters (a, b, c, d, e) indicate significant differences at P values of
    Figure Legend Snippet: The HR-like response correlates with the silencing suppression activity of P25. (A) Leaves of N. benthamiana were infiltrated with GUS alone or combinations of Agrobacterium cultures containing binary constructs expressing a free GFP reporter gene plus T7-tagged versions of either P25wt or P25 A104V, T117A, K124E mutants or P25stop mutant, as indicated. Photographs were taken with long-wavelength UV light at 3 days postagroinoculation (dpa) (upper panels). Total RNA (10 μg) extracted from infiltrated tissues was hybridized with a probe complementary to GFP (lower panel). Ethidium bromide staining of 25S rRNA is shown as a loading control. (B) Western blot analysis of extracts derived from leaf patches infiltrated with combinations of PPV HCwt plus either P25 wt, A104V, T117A, K124E, P25stop, or GUS at 6 dpa, using antibodies against the T7 epitope. The lower panel shows the Ponceau S-stained membrane after blotting, as a loading control. (C) Leaves were infiltrated with GUS alone or combinations of Agrobacterium cultures containing PPV HCwt plus either T7-tagged versions of P25 wt, A104V, T117A, K124E, P25stop, or GUS. Leaves were stained with DAB solution at 9 dpa. (D) Electrolyte leakage from leaf disks infiltrated with T7-tagged versions of P25 wt, A104V, T117A, K124E, or P25stop, either alone or in combination with PPV HCwt at 9 dpa. GUS alone was infiltrated as a control. Data represent the means ± standard errors for 18 replicates, each consisting of four plants that received the same treatment in three independent experiments. Statistically significant differences between means were determined by employing Scheffé's multiple-range test. Different letters (a, b, c, d, e) indicate significant differences at P values of

    Techniques Used: Activity Assay, Construct, Expressing, Mutagenesis, Staining, Western Blot, Derivative Assay

    8) Product Images from "The Small GTPase Rap1 Is a Novel Regulator of RPE Cell Barrier Function"

    Article Title: The Small GTPase Rap1 Is a Novel Regulator of RPE Cell Barrier Function

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.11-7295

    Loss of Rap1 GTPase activity by expressing RapGAP reduces plateau TER in ARPE-19 cells. ( A ) Comparison of active (GTP-bound) Rap1 in lysates of ARPE-19 cells expressing either GFP control or RapGAP and assayed. Active (GTP-bound) pool of Rap1 is shown
    Figure Legend Snippet: Loss of Rap1 GTPase activity by expressing RapGAP reduces plateau TER in ARPE-19 cells. ( A ) Comparison of active (GTP-bound) Rap1 in lysates of ARPE-19 cells expressing either GFP control or RapGAP and assayed. Active (GTP-bound) pool of Rap1 is shown

    Techniques Used: Activity Assay, Expressing

    Electrical impedance of the RPE cell monolayer is decreased on expression of RapGAP. ARPE-19 cells expressing either GFP or RapGAP were plated at equal cell density into microelectrode-coated wells of an E-Plate 16 and electrical impedance was measured
    Figure Legend Snippet: Electrical impedance of the RPE cell monolayer is decreased on expression of RapGAP. ARPE-19 cells expressing either GFP or RapGAP were plated at equal cell density into microelectrode-coated wells of an E-Plate 16 and electrical impedance was measured

    Techniques Used: Expressing

    9) Product Images from "Zinc transporter 2 (SLC30A2) can suppress the vesicular zinc defect of adaptor protein 3-depleted fibroblasts by promoting zinc accumulation in lysosomes"

    Article Title: Zinc transporter 2 (SLC30A2) can suppress the vesicular zinc defect of adaptor protein 3-depleted fibroblasts by promoting zinc accumulation in lysosomes

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2007.02.006

    Overexpression of GFP-tagged ZnT2 restores chelatable zinc stores in AP-3-depleted cells. (a-c) Cultured M1 fibroblasts were treated with control siRNA or with a siRNA duplex specifically designed to target the δ subunit of the AP-3 protein complex.
    Figure Legend Snippet: Overexpression of GFP-tagged ZnT2 restores chelatable zinc stores in AP-3-depleted cells. (a-c) Cultured M1 fibroblasts were treated with control siRNA or with a siRNA duplex specifically designed to target the δ subunit of the AP-3 protein complex.

    Techniques Used: Over Expression, Cell Culture

    Localization of GFP-tagged ZnT2, ZnT3 and ZnT4 proteins to late endocytic organelles of stably transfected M1 fibroblasts. (a-i) Stable M1 fibroblast lines expressing human ZnT2 (a-c), ZnT3 (d-f) or ZnT4 (g-i) fused at their N-terminus to GFP were subjected
    Figure Legend Snippet: Localization of GFP-tagged ZnT2, ZnT3 and ZnT4 proteins to late endocytic organelles of stably transfected M1 fibroblasts. (a-i) Stable M1 fibroblast lines expressing human ZnT2 (a-c), ZnT3 (d-f) or ZnT4 (g-i) fused at their N-terminus to GFP were subjected

    Techniques Used: Stable Transfection, Transfection, Expressing

    Effects of the expression of GFP-tagged ZnT2, ZnT3 and ZnT4 proteins on the accumulation of chelatable zinc by human M1 fibroblasts. (a-f) Stable M1 fibroblasts expressing ZnT2 tagged with GFP at its aminoterminus (a-c) or at its carboxyl-terminus (d-f)
    Figure Legend Snippet: Effects of the expression of GFP-tagged ZnT2, ZnT3 and ZnT4 proteins on the accumulation of chelatable zinc by human M1 fibroblasts. (a-f) Stable M1 fibroblasts expressing ZnT2 tagged with GFP at its aminoterminus (a-c) or at its carboxyl-terminus (d-f)

    Techniques Used: Expressing

    Analysis of the colocalization of GFP-tagged ZnT2, ZnT3 and ZnT4 proteins with chelatable zinc stores in M1 cells. (a-l) Human M1 fibroblasts stably expressing ZnT2, ZnT3 or ZnT4 GFP-tagged at the N-terminus (GFP-ZnT2 and GFP-ZnT3) or C-terminus (ZnT2-GFP
    Figure Legend Snippet: Analysis of the colocalization of GFP-tagged ZnT2, ZnT3 and ZnT4 proteins with chelatable zinc stores in M1 cells. (a-l) Human M1 fibroblasts stably expressing ZnT2, ZnT3 or ZnT4 GFP-tagged at the N-terminus (GFP-ZnT2 and GFP-ZnT3) or C-terminus (ZnT2-GFP

    Techniques Used: Stable Transfection, Expressing

    Overexpression of GFP-ZnT2 elicits a shift in the distribution of chelatable zinc to mature lysosomes. (a-h) M1 fibroblasts were transiently transfected with a plasmid encoding the GFP-ZnT2 fusion protein, loaded with Texas-Red-conjugated dextran to label
    Figure Legend Snippet: Overexpression of GFP-ZnT2 elicits a shift in the distribution of chelatable zinc to mature lysosomes. (a-h) M1 fibroblasts were transiently transfected with a plasmid encoding the GFP-ZnT2 fusion protein, loaded with Texas-Red-conjugated dextran to label

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    10) Product Images from "Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿"

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01155-06

    AUF1 p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 mRNA bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.
    Figure Legend Snippet: AUF1 p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 mRNA bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.

    Techniques Used: Construct, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    Effect of the proteasome inhibitor MG132 on IL-6 mRNA and AUF1 stability. (A) The decay of GFP-IL-6_wt mRNA in mouse NIH 3T3 cells was analyzed using the Tet-Off system and real-time PCR after a treatment for 4 h with 40 μM MG132. Average results ± SD of four experiments are shown, and half-lives were calculated by linear regression of semilogarithmic plots. (B) Exogenous myc-tagged AUF1 p37 was induced with mifepristone in NIH 3T3 cells and its level analyzed on a Western blot before and after treatment for 4 h with 40 μM MG132. Expression of γ-tubulin was unchanged by MG132 and served as a loading control.
    Figure Legend Snippet: Effect of the proteasome inhibitor MG132 on IL-6 mRNA and AUF1 stability. (A) The decay of GFP-IL-6_wt mRNA in mouse NIH 3T3 cells was analyzed using the Tet-Off system and real-time PCR after a treatment for 4 h with 40 μM MG132. Average results ± SD of four experiments are shown, and half-lives were calculated by linear regression of semilogarithmic plots. (B) Exogenous myc-tagged AUF1 p37 was induced with mifepristone in NIH 3T3 cells and its level analyzed on a Western blot before and after treatment for 4 h with 40 μM MG132. Expression of γ-tubulin was unchanged by MG132 and served as a loading control.

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

    11) Product Images from "The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)"

    Article Title: The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

    Journal: Molecular Biology of the Cell

    doi:

    (A) Yrb1p is a major Xpo1p-binding protein. Xpo1p-ZZ was purified from yeast extracts in the presence of recombinant Gsp1pQ71L-GTP as described in Materials and Methods, and proteins bound to immobilized Xpo1p were eluted with 500 mM KCl. The input (I), flow-through (FT), and eluate (E) were analyzed by SDS PAGE and Coomassie blue staining. The two major proteins in the eluate were identified by MALDI and Western blotting and correspond to Gsp1p and Yrb1p. Molecular weight markers (M) are in kDa. (B-E) Yrb1p shuttles between the cytoplasm and the nucleus. Wild-type cells (B) or xpo1–1 cells (C-E) expressing GFP-YRB1 were grown in liquid medium at 25°C. The cultures were kept at 25°C (C), shifted to 37°C for 2 h (D), or shifted to 37°C for 2 h and then shifted back to 25°C for 2 h (E). To inhibit protein synthesis, cycloheximide (final concentration 0.1 mg/ml) was added to the cultures before the temperature shift. The cells were viewed by fluorescence microscopy to visualize GFP-Yrb1p. The perinuclear staining in wild-type cells is indicated by arrows. (F-K) Yrb1p has two nuclear targeting sequences. Wild-type cells (F, H, and J) or xpo1–1 mutants (G, I, and K) were transformed with plasmids encoding GFP-Yrb1p, GFP-Yrb1ΔN62p, or GFP-Yrb1p 1–40, as indicated. The cultures were incubated at 30°C ( XPO1 cells) or 25°C ( xpo1–1 cells) in raffinose-containing medium. Expression of the GFP fusions was induced by 2% galactose and repressed by addition of 2% glucose after 1 h. Cells were kept at 30°C ( XPO1 ) or shifted to 37°C for 2 h ( xpo1–1 ) and viewed by fluorescence microscopy.
    Figure Legend Snippet: (A) Yrb1p is a major Xpo1p-binding protein. Xpo1p-ZZ was purified from yeast extracts in the presence of recombinant Gsp1pQ71L-GTP as described in Materials and Methods, and proteins bound to immobilized Xpo1p were eluted with 500 mM KCl. The input (I), flow-through (FT), and eluate (E) were analyzed by SDS PAGE and Coomassie blue staining. The two major proteins in the eluate were identified by MALDI and Western blotting and correspond to Gsp1p and Yrb1p. Molecular weight markers (M) are in kDa. (B-E) Yrb1p shuttles between the cytoplasm and the nucleus. Wild-type cells (B) or xpo1–1 cells (C-E) expressing GFP-YRB1 were grown in liquid medium at 25°C. The cultures were kept at 25°C (C), shifted to 37°C for 2 h (D), or shifted to 37°C for 2 h and then shifted back to 25°C for 2 h (E). To inhibit protein synthesis, cycloheximide (final concentration 0.1 mg/ml) was added to the cultures before the temperature shift. The cells were viewed by fluorescence microscopy to visualize GFP-Yrb1p. The perinuclear staining in wild-type cells is indicated by arrows. (F-K) Yrb1p has two nuclear targeting sequences. Wild-type cells (F, H, and J) or xpo1–1 mutants (G, I, and K) were transformed with plasmids encoding GFP-Yrb1p, GFP-Yrb1ΔN62p, or GFP-Yrb1p 1–40, as indicated. The cultures were incubated at 30°C ( XPO1 cells) or 25°C ( xpo1–1 cells) in raffinose-containing medium. Expression of the GFP fusions was induced by 2% galactose and repressed by addition of 2% glucose after 1 h. Cells were kept at 30°C ( XPO1 ) or shifted to 37°C for 2 h ( xpo1–1 ) and viewed by fluorescence microscopy.

    Techniques Used: Binding Assay, Purification, Recombinant, Flow Cytometry, SDS Page, Staining, Western Blot, Molecular Weight, Expressing, Concentration Assay, Fluorescence, Microscopy, Transformation Assay, Incubation

    12) Product Images from "The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton ▿"

    Article Title: The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton ▿

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00112-10

    Schematic representation of constructs. FL, full-length PDEB1 or PDEB2; open circle, C-terminal c-Myc or HA tag; B1(1–212)::GFP, B1(1–114)::GFP, and B1(1–70)::GFP, GFP fusions carrying the N-terminal 212, 114, and 70 amino acids
    Figure Legend Snippet: Schematic representation of constructs. FL, full-length PDEB1 or PDEB2; open circle, C-terminal c-Myc or HA tag; B1(1–212)::GFP, B1(1–114)::GFP, and B1(1–70)::GFP, GFP fusions carrying the N-terminal 212, 114, and 70 amino acids

    Techniques Used: Construct

    The N terminus of PDEB1, but not that of PDEB2, confers integration into the flagellar skeleton. (A) Amino acids 1 to 212 of PDEB1 lead to stable integration of the GFP reporter into the flagellar skeleton; (B) amino acids 1 to 212 of PDEB2 do not; (C)
    Figure Legend Snippet: The N terminus of PDEB1, but not that of PDEB2, confers integration into the flagellar skeleton. (A) Amino acids 1 to 212 of PDEB1 lead to stable integration of the GFP reporter into the flagellar skeleton; (B) amino acids 1 to 212 of PDEB2 do not; (C)

    Techniques Used:

    13) Product Images from "Dynamic association of calcium channel subunits at the cellular membrane"

    Article Title: Dynamic association of calcium channel subunits at the cellular membrane

    Journal: Neurophotonics

    doi: 10.1117/1.NPh.3.4.041809

    Dynamics of channel subunits in the neuronal membrane. (a) Example for a FRAP experiment of Ca V 2.2 ∷ GFP intra channels expressed in hippocampal neurons 14 DIV, scale bare indicate 5 μ m. (b) Example recovery curve of the region indicated in (a). The mean fluorescent recovery for Ca V 2.2 ∷ GFP intra channels was 24.8%±3% (data from 30 clusters analyzed out of 2 independent cultures). (c) Example traces for α 2 δ 1 ∷ HA-subunits labeled with QDs as indicated in the sketch. (d) Medians of diffusion coefficients for GPI::GFP, α 2 δ 1 ∷ HA-subunit alone, and α 2 δ 1 ∷ HA-subunit coexpressed with Ca V 2.2 ∷ GFP intra channels in axonal and presynaptic membranes, as indicated. The number of trajectories is given for each condition and data are out of two independent experiments. The significance was tested by a Kruskal–Wallis test followed by a Dunns test to compare individual columns.
    Figure Legend Snippet: Dynamics of channel subunits in the neuronal membrane. (a) Example for a FRAP experiment of Ca V 2.2 ∷ GFP intra channels expressed in hippocampal neurons 14 DIV, scale bare indicate 5 μ m. (b) Example recovery curve of the region indicated in (a). The mean fluorescent recovery for Ca V 2.2 ∷ GFP intra channels was 24.8%±3% (data from 30 clusters analyzed out of 2 independent cultures). (c) Example traces for α 2 δ 1 ∷ HA-subunits labeled with QDs as indicated in the sketch. (d) Medians of diffusion coefficients for GPI::GFP, α 2 δ 1 ∷ HA-subunit alone, and α 2 δ 1 ∷ HA-subunit coexpressed with Ca V 2.2 ∷ GFP intra channels in axonal and presynaptic membranes, as indicated. The number of trajectories is given for each condition and data are out of two independent experiments. The significance was tested by a Kruskal–Wallis test followed by a Dunns test to compare individual columns.

    Techniques Used: Labeling, Diffusion-based Assay

    14) Product Images from "Generation of specific inhibitors of SUMO1- and SUMO2/3-mediated protein-protein interactions using Affimer (Adhiron) technology 1"

    Article Title: Generation of specific inhibitors of SUMO1- and SUMO2/3-mediated protein-protein interactions using Affimer (Adhiron) technology 1

    Journal: Science signaling

    doi: 10.1126/scisignal.aaj2005

    S-Affs do not inhibit SUMO conjugation or deconjugation. (A, B) In vitro SUMOylation in the presence of the indicated S-Affs for SUMO-1 (A) or SUMO-2 (B) conjugation to GST-tagged RanGAP1 fragment (RG1; amino acids 418-587 encompassing a consensus SUMOylation site). Experiments were performed in the presence of increasing concentrations of S-Aff (0.1:1, 1:1, or 10:1 ratios of S-Aff:SUMO). GFP-Adh is a control Affimer raised against green fluorescent protein (GFP) and does not bind to SUMO. Blots were probed with an antibody recognizing GFP. (C) SUMOylation of FLAG-tagged PML-I in HEK293T also expressing the indicated FLAG-tagged S-Affs. SUMOylation was induced with arsenic trioxide (As 2 O 3 ). Proteins were detected with the antibody recognizing the FLAG tag or with an antibody recognizing GAPDH as a loading control. (D, E) Coomassie-stained SDS-PAGE showing in vitro assays to assess SUMO-1 (D) or SUMO-2 (E) de-SUMOylation by the SUMO deconjugases SENP1 and SENP2. All data are representative of at least 2 independent experiments.
    Figure Legend Snippet: S-Affs do not inhibit SUMO conjugation or deconjugation. (A, B) In vitro SUMOylation in the presence of the indicated S-Affs for SUMO-1 (A) or SUMO-2 (B) conjugation to GST-tagged RanGAP1 fragment (RG1; amino acids 418-587 encompassing a consensus SUMOylation site). Experiments were performed in the presence of increasing concentrations of S-Aff (0.1:1, 1:1, or 10:1 ratios of S-Aff:SUMO). GFP-Adh is a control Affimer raised against green fluorescent protein (GFP) and does not bind to SUMO. Blots were probed with an antibody recognizing GFP. (C) SUMOylation of FLAG-tagged PML-I in HEK293T also expressing the indicated FLAG-tagged S-Affs. SUMOylation was induced with arsenic trioxide (As 2 O 3 ). Proteins were detected with the antibody recognizing the FLAG tag or with an antibody recognizing GAPDH as a loading control. (D, E) Coomassie-stained SDS-PAGE showing in vitro assays to assess SUMO-1 (D) or SUMO-2 (E) de-SUMOylation by the SUMO deconjugases SENP1 and SENP2. All data are representative of at least 2 independent experiments.

    Techniques Used: Conjugation Assay, In Vitro, Expressing, FLAG-tag, Staining, SDS Page

    Isoform specificity of S-Affs binding to SUMO in vitro. ( A ) Immunoprecipitation of GFP-tagged SUMO-1 or SUMO-2 expressed in HEK293T cells with the indicated FLAG-tagged S-Aff or in cells transfected with only the vector for the S-Aff. Proteins were immunoprecipitated (IP) with an antibody recognizing GFP and immunoblotted (IB) for either the FLAG tag or GFP. The left blot shows the coimmunoprecipitation results, and the right blot shows the proteins in the lysate prior to immunoprecipitation (Input). (B) Immunoblot showing pulldown of purified (bacterially-expressed) His-tagged S-Affs following incubation with extracts from HEK293T cells expressing GFP, GFP-tagged SUMO-1, or GFP-tagged SUMO-2S-Aff. Overexpressed proteins that interacted with His-S-Affs (top) and input samples taken prior to incubating with His-S-Affs (bottom) were detected with antibodies recognizing GFP. Pulled-down His-S-Affs were detected by Coomassie staining (middle panel). Data are representative of 2 experiments.
    Figure Legend Snippet: Isoform specificity of S-Affs binding to SUMO in vitro. ( A ) Immunoprecipitation of GFP-tagged SUMO-1 or SUMO-2 expressed in HEK293T cells with the indicated FLAG-tagged S-Aff or in cells transfected with only the vector for the S-Aff. Proteins were immunoprecipitated (IP) with an antibody recognizing GFP and immunoblotted (IB) for either the FLAG tag or GFP. The left blot shows the coimmunoprecipitation results, and the right blot shows the proteins in the lysate prior to immunoprecipitation (Input). (B) Immunoblot showing pulldown of purified (bacterially-expressed) His-tagged S-Affs following incubation with extracts from HEK293T cells expressing GFP, GFP-tagged SUMO-1, or GFP-tagged SUMO-2S-Aff. Overexpressed proteins that interacted with His-S-Affs (top) and input samples taken prior to incubating with His-S-Affs (bottom) were detected with antibodies recognizing GFP. Pulled-down His-S-Affs were detected by Coomassie staining (middle panel). Data are representative of 2 experiments.

    Techniques Used: Binding Assay, In Vitro, Immunoprecipitation, Transfection, Plasmid Preparation, FLAG-tag, Purification, Incubation, Expressing, Staining

    15) Product Images from "Dynein-mediated transport and membrane trafficking control PAR3 polarised distribution"

    Article Title: Dynein-mediated transport and membrane trafficking control PAR3 polarised distribution

    Journal: eLife

    doi: 10.7554/eLife.40212

    PAR3 asymmetry depends on RAB5. ( A ) PAR3-GFP ( A’ ), ( A’’’ green) expressed in germline is present occasionally in RAB5-positive early endosomes ( A’’ ), ( A’’’ ) magenta). A’, A’’ and A’’’ are magnifications of A (white frame). Arrowheads show the vesicles that are associated with PAR3 and RAB5. ( B–F ) PAR3 distribution in response to RAB5 activity impairment. PAR3-GFP is expressed in germinal cells at stage 9B in RAB5 RNAi, RAB5DN (S43N) or in mCherry knockdown (control) contexts. ( B–D ) Representative images of PAR3 distribution under control conditions ( B ), in RAB5 RNAi ( C ), or in RAB5DN (S43N) ( D ). Scale bars indicate 30 µm. ( E ) Quantification of PAR3 density at each plasma membrane domain. Error bars indicate SEM. ( F ) Quantification of PAR3 posterior exclusion in different RAB5 mutant contexts. Control (stage 9B, n = 10); RAB5 RNAi (stage 9B, n = 12); RAB5DN (S43N) (stage 9B, n = 6). Mann-Whitney test, ns: not significant; *p
    Figure Legend Snippet: PAR3 asymmetry depends on RAB5. ( A ) PAR3-GFP ( A’ ), ( A’’’ green) expressed in germline is present occasionally in RAB5-positive early endosomes ( A’’ ), ( A’’’ ) magenta). A’, A’’ and A’’’ are magnifications of A (white frame). Arrowheads show the vesicles that are associated with PAR3 and RAB5. ( B–F ) PAR3 distribution in response to RAB5 activity impairment. PAR3-GFP is expressed in germinal cells at stage 9B in RAB5 RNAi, RAB5DN (S43N) or in mCherry knockdown (control) contexts. ( B–D ) Representative images of PAR3 distribution under control conditions ( B ), in RAB5 RNAi ( C ), or in RAB5DN (S43N) ( D ). Scale bars indicate 30 µm. ( E ) Quantification of PAR3 density at each plasma membrane domain. Error bars indicate SEM. ( F ) Quantification of PAR3 posterior exclusion in different RAB5 mutant contexts. Control (stage 9B, n = 10); RAB5 RNAi (stage 9B, n = 12); RAB5DN (S43N) (stage 9B, n = 6). Mann-Whitney test, ns: not significant; *p

    Techniques Used: Activity Assay, Mutagenesis, MANN-WHITNEY

    Dynamic PAR3 distribution along the oocyte anterior posterior axis. ( A ) Description of the quantification Fiji Macro. After selecting three points in the oocyte (yellow stars) and delimitation of oocyte perimeter, the macro allows us to obtain different data about protein repartition in oocytes: the intensity profile of the plasma membrane (magenta); the mean fluorescent intensity and the length of each of the plasma membrane domainsare automatically generated (anterior/APM in red; lateral/LPM in green; posterior/PPM in blue) and the mean signal intensity inside the cytoplasm (cyan). ( B–E ) Distribution of PAR3 between stages 8 and 10 in representative examples. ( B ) Localisation of PAR3-GFP, expressed in the germline under control of the maternal driver Tub67c-GAL4, from stage 8 to stage 10. The brackets indicate oocytes. ( C ) Representative intensity profiles of plasma membrane distribution in a single oocyte (APM in red, PPM in blue). Green arrows highlight PAR3 posterior accumulation, and red arrows PAR3 posterior exclusion. ( D ) Raw quantity of PAR3 in each plasma membrane domain from stage 8 to stage 10 (APM in red; LPM in green; PPM in blue). ( E ) To avoid size/expression fluctuations of oocytes between the different mutant genotypes, PAR3 distribution has been normalised by the length of the membrane and the total oocyte signal (density/total). In this case, we cannot compare the level between the different stages but only the asymmetrical distribution of PAR3 between the different domains. ( F ) Evolution of PAR3 asymmetry from stage 8 to stage 10. The asymmetry ratio (APM/PPM of PAR3 density) highlights the increase of PAR3 polarity in oocytes from stage 8 to stage 10. Stage 8, n = 8; stage 9A, n = 9; stage 9B, n = 15; stage 10, n = 14. Mann-Whitney test, NS: not significant; **: p
    Figure Legend Snippet: Dynamic PAR3 distribution along the oocyte anterior posterior axis. ( A ) Description of the quantification Fiji Macro. After selecting three points in the oocyte (yellow stars) and delimitation of oocyte perimeter, the macro allows us to obtain different data about protein repartition in oocytes: the intensity profile of the plasma membrane (magenta); the mean fluorescent intensity and the length of each of the plasma membrane domainsare automatically generated (anterior/APM in red; lateral/LPM in green; posterior/PPM in blue) and the mean signal intensity inside the cytoplasm (cyan). ( B–E ) Distribution of PAR3 between stages 8 and 10 in representative examples. ( B ) Localisation of PAR3-GFP, expressed in the germline under control of the maternal driver Tub67c-GAL4, from stage 8 to stage 10. The brackets indicate oocytes. ( C ) Representative intensity profiles of plasma membrane distribution in a single oocyte (APM in red, PPM in blue). Green arrows highlight PAR3 posterior accumulation, and red arrows PAR3 posterior exclusion. ( D ) Raw quantity of PAR3 in each plasma membrane domain from stage 8 to stage 10 (APM in red; LPM in green; PPM in blue). ( E ) To avoid size/expression fluctuations of oocytes between the different mutant genotypes, PAR3 distribution has been normalised by the length of the membrane and the total oocyte signal (density/total). In this case, we cannot compare the level between the different stages but only the asymmetrical distribution of PAR3 between the different domains. ( F ) Evolution of PAR3 asymmetry from stage 8 to stage 10. The asymmetry ratio (APM/PPM of PAR3 density) highlights the increase of PAR3 polarity in oocytes from stage 8 to stage 10. Stage 8, n = 8; stage 9A, n = 9; stage 9B, n = 15; stage 10, n = 14. Mann-Whitney test, NS: not significant; **: p

    Techniques Used: Generated, Expressing, Mutagenesis, MANN-WHITNEY

    Role of RAB11 on PAR3 asymmetrical localisation. ( A ) Colocalisation of PAR3 (PAR3-GFP maternally expressed) with vesicular trafficking markers. To quantify the colocalisation, we measured the Pearson’s coefficient on stage 9B oocytes and results are presented in the box plot. As a control, we used the value of colocalisation obtained with the same images but after rotation of one by 90 degrees. The stars represent the p-value with the control. For HRS, KDEL, and SYX16: n = 7; for PH PLC : n = 8; for RAB5: n = 10; for RAB11: n = 13. ( B ) PAR3-GFP ( B’ ), green) expressed in germline is present occasionally in RAB11-positive recycling endosomes ( B’’ ), ( B’’’ ) magenta). B’ , B’’ , and B’’’ are magnifications of B (white frame). Arrowheads show the vesicles associated with PAR3 and RAB11. The arrow points to the vesicle associated only with PAR3. ( C,D ) RAB11 effect on asymmetrical distribution of PAR3. The antero-posterior asymmetry ( C ) and the posterior exclusion ( D ) of PAR3 at stage 9B have been evaluated in rab11 P2148 germline clones. Control (stage 9B, n = 10); rab11 P2148 (stage 9B, n = 10). Mann-Whitney test, NS: not significant; *p
    Figure Legend Snippet: Role of RAB11 on PAR3 asymmetrical localisation. ( A ) Colocalisation of PAR3 (PAR3-GFP maternally expressed) with vesicular trafficking markers. To quantify the colocalisation, we measured the Pearson’s coefficient on stage 9B oocytes and results are presented in the box plot. As a control, we used the value of colocalisation obtained with the same images but after rotation of one by 90 degrees. The stars represent the p-value with the control. For HRS, KDEL, and SYX16: n = 7; for PH PLC : n = 8; for RAB5: n = 10; for RAB11: n = 13. ( B ) PAR3-GFP ( B’ ), green) expressed in germline is present occasionally in RAB11-positive recycling endosomes ( B’’ ), ( B’’’ ) magenta). B’ , B’’ , and B’’’ are magnifications of B (white frame). Arrowheads show the vesicles associated with PAR3 and RAB11. The arrow points to the vesicle associated only with PAR3. ( C,D ) RAB11 effect on asymmetrical distribution of PAR3. The antero-posterior asymmetry ( C ) and the posterior exclusion ( D ) of PAR3 at stage 9B have been evaluated in rab11 P2148 germline clones. Control (stage 9B, n = 10); rab11 P2148 (stage 9B, n = 10). Mann-Whitney test, NS: not significant; *p

    Techniques Used: Planar Chromatography, Clone Assay, MANN-WHITNEY

    PAR3 recovery after APM photobleaching. ( A ) PAR3-GFP expressed in ovarian follicle ( Tub67c-GAL4; UASp PAR3-GFP ) is photobleached in all the nurse cells and at the APM (yellow area, A1 ). The fluorescence recovery was followed for around 1400 s ( A2 ). ( B ) PAR3 quantity in each domain (anterior and posterior domains) was quantified using the same method as previously for three ovarian follicles and raised to 0 after bleaching. It can be seen that, after photobleaching, PAR3 accumulates progressively at the anterior while it is excluded from the posterior. ( C ) PAR3 quantity of each zone before FRAP was normalised to 1, and recovery of fluorescence was observed. ( D ) The same experiment as in ( A ) was performed on ovarian follicle incubated with colcemid. The quantification is shown in graphs in ( C ) and ( D ). In ( B ) and ( C ), the error bar represents SEM. The scale bars represent 20 µm.
    Figure Legend Snippet: PAR3 recovery after APM photobleaching. ( A ) PAR3-GFP expressed in ovarian follicle ( Tub67c-GAL4; UASp PAR3-GFP ) is photobleached in all the nurse cells and at the APM (yellow area, A1 ). The fluorescence recovery was followed for around 1400 s ( A2 ). ( B ) PAR3 quantity in each domain (anterior and posterior domains) was quantified using the same method as previously for three ovarian follicles and raised to 0 after bleaching. It can be seen that, after photobleaching, PAR3 accumulates progressively at the anterior while it is excluded from the posterior. ( C ) PAR3 quantity of each zone before FRAP was normalised to 1, and recovery of fluorescence was observed. ( D ) The same experiment as in ( A ) was performed on ovarian follicle incubated with colcemid. The quantification is shown in graphs in ( C ) and ( D ). In ( B ) and ( C ), the error bar represents SEM. The scale bars represent 20 µm.

    Techniques Used: Fluorescence, Incubation

    IKKε regulates PAR3 microtubule unloading and APM accumulation. ( A–C ) Distribution of PAR3 in response to IKK ε knockdown. ( A ) IKK ε knockdown affects the localisation of PAR3 and leads to an accumulation of circular actin clumps (ACs) enriched in PAR3. ( B ) Quantification of PAR3 density at each plasma membrane domain from stage 9A to stage 10 oocytes in WT or IKK ε knockdown contexts ( Tub67c-GAL4 ; UASp RNAi ikk ε; UASp PAR3-GFP ). ( C ) Quantification of PAR3-GFP distribution in the cytoplasm related to the whole oocyte intensity at stage 9B in WT or in IKK ε knockdown contexts. For ( B, C ), Control (stage 9A, n = 9; stage 9B, n = 15; stage 10, n = 14); RNAi IKKε (stage 9A, n = 15; stage 9B, n = 10; stage 10, n = 9). ( C ) Mann-Whitney test. Error bars indicate SEM. **** indicates p
    Figure Legend Snippet: IKKε regulates PAR3 microtubule unloading and APM accumulation. ( A–C ) Distribution of PAR3 in response to IKK ε knockdown. ( A ) IKK ε knockdown affects the localisation of PAR3 and leads to an accumulation of circular actin clumps (ACs) enriched in PAR3. ( B ) Quantification of PAR3 density at each plasma membrane domain from stage 9A to stage 10 oocytes in WT or IKK ε knockdown contexts ( Tub67c-GAL4 ; UASp RNAi ikk ε; UASp PAR3-GFP ). ( C ) Quantification of PAR3-GFP distribution in the cytoplasm related to the whole oocyte intensity at stage 9B in WT or in IKK ε knockdown contexts. For ( B, C ), Control (stage 9A, n = 9; stage 9B, n = 15; stage 10, n = 14); RNAi IKKε (stage 9A, n = 15; stage 9B, n = 10; stage 10, n = 9). ( C ) Mann-Whitney test. Error bars indicate SEM. **** indicates p

    Techniques Used: MANN-WHITNEY

    SKTL-dependent PAR3 posterior exclusion bypasses regulation by PAR1. ( A–C ) Representative distribution of PAR3-GFP in stage 9B oocyte in a control situation ( A ), in RNAi PAR1 context ( B ), or when PAR3 phosphorylation sites by PAR1 are mutated ( C ). In the control genotype, PAR3 is excluded from PPM (arrow), unlike the other two genotypes. N indicates the oocyte nucleus position. The scale bars represent 30 µm. ( D ) PAR3 posterior exclusion in response to PAR1 at stage 9B. In germinal cells, PAR3AA-GFP, a mutant form, non phosphorylable by PAR1 or PAR3-GFP is expressed with nothing, with par1, or with mCherry knock-down contexts. The posterior exclusion ratios in stage 9B oocytes are represented. PAR3 (stage 9B, n = 15); PAR3-AA (stage 9B, n = 6); PAR3 RNAi mCherry (stage 9B, n = 10); PAR3 RNAi PAR1 (stage 9B, n = 10). ( E ) SKTL effect on PAR3 posterior exclusion is observed in combination with a PAR1 activity decrease (in green) or with the PAR3-AA non phosphorylable form (in red). PAR3, RNAi PAR1, RNAi mCherry (stage 9B, n = 10); PAR3, RNAi PAR1, mycSKTL (stage 9B, n = 11) PAR3 RNAi PAR1 (stage 9B, n = 10); PAR3, RNAi mCherry, mycSKTL (stage 9B, n = 10); PAR3-AA, RNAi mCherry (stage 9B, n = 10); PAR3-AA, mycSKTL (stage 9B, n = 10). Mann-Whitney test, NS: not significant; *p
    Figure Legend Snippet: SKTL-dependent PAR3 posterior exclusion bypasses regulation by PAR1. ( A–C ) Representative distribution of PAR3-GFP in stage 9B oocyte in a control situation ( A ), in RNAi PAR1 context ( B ), or when PAR3 phosphorylation sites by PAR1 are mutated ( C ). In the control genotype, PAR3 is excluded from PPM (arrow), unlike the other two genotypes. N indicates the oocyte nucleus position. The scale bars represent 30 µm. ( D ) PAR3 posterior exclusion in response to PAR1 at stage 9B. In germinal cells, PAR3AA-GFP, a mutant form, non phosphorylable by PAR1 or PAR3-GFP is expressed with nothing, with par1, or with mCherry knock-down contexts. The posterior exclusion ratios in stage 9B oocytes are represented. PAR3 (stage 9B, n = 15); PAR3-AA (stage 9B, n = 6); PAR3 RNAi mCherry (stage 9B, n = 10); PAR3 RNAi PAR1 (stage 9B, n = 10). ( E ) SKTL effect on PAR3 posterior exclusion is observed in combination with a PAR1 activity decrease (in green) or with the PAR3-AA non phosphorylable form (in red). PAR3, RNAi PAR1, RNAi mCherry (stage 9B, n = 10); PAR3, RNAi PAR1, mycSKTL (stage 9B, n = 11) PAR3 RNAi PAR1 (stage 9B, n = 10); PAR3, RNAi mCherry, mycSKTL (stage 9B, n = 10); PAR3-AA, RNAi mCherry (stage 9B, n = 10); PAR3-AA, mycSKTL (stage 9B, n = 10). Mann-Whitney test, NS: not significant; *p

    Techniques Used: Mutagenesis, Activity Assay, MANN-WHITNEY

    By producing PI(4,5)P2, SKTL controls PAR3 APM accumulation and PPM exclusion. ( A–F ) Distribution of PAR3 in response to SKTL activity. PAR3-GFP is expressed in germinal cells at stage 9B under control conditions, or with overexpression (OE) of Myc-SKTL, Myc-SKTL DNRQ , or in context sktl 2.3 / sktl ∆5 . ( A–C ) Representative distribution of PAR3-GFP in oocytes in these different genetic contexts. ( D ) Quantification of PAR3 density at each plasma membrane domain in sktl mutant or SKTL overexpressed (OE) contexts in stage 9B oocytes. Error bars indicate SEM. ( E ) Antero-posterior asymmetry of PAR3 (ratio APM/PPM density) at stage 9B in control or sktl mutant. ( F ) Quantification of PAR3 posterior exclusion ratio in sktl mutant or SKTL overexpressed contexts at stage 9B. For ( D–F ): control (stage 9B, n = 10); sktl 2.3 / sktl ∆5 (stage 9B, n = 8); Myc-SKTL (stage 9B, n = 8); Myc-SKTL DNRQ (stage 9B, n = 10). Mann-Whitney test, NS: not significant; *p
    Figure Legend Snippet: By producing PI(4,5)P2, SKTL controls PAR3 APM accumulation and PPM exclusion. ( A–F ) Distribution of PAR3 in response to SKTL activity. PAR3-GFP is expressed in germinal cells at stage 9B under control conditions, or with overexpression (OE) of Myc-SKTL, Myc-SKTL DNRQ , or in context sktl 2.3 / sktl ∆5 . ( A–C ) Representative distribution of PAR3-GFP in oocytes in these different genetic contexts. ( D ) Quantification of PAR3 density at each plasma membrane domain in sktl mutant or SKTL overexpressed (OE) contexts in stage 9B oocytes. Error bars indicate SEM. ( E ) Antero-posterior asymmetry of PAR3 (ratio APM/PPM density) at stage 9B in control or sktl mutant. ( F ) Quantification of PAR3 posterior exclusion ratio in sktl mutant or SKTL overexpressed contexts at stage 9B. For ( D–F ): control (stage 9B, n = 10); sktl 2.3 / sktl ∆5 (stage 9B, n = 8); Myc-SKTL (stage 9B, n = 8); Myc-SKTL DNRQ (stage 9B, n = 10). Mann-Whitney test, NS: not significant; *p

    Techniques Used: Activity Assay, Over Expression, Mutagenesis, MANN-WHITNEY

    16) Product Images from "A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus"

    Article Title: A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12532

    Cell‐to‐cell movement of Barley mosaic virus (BaMV) in Luc ‐ and NbTRXh2 ‐knockdown plants. (A) Areas of green fluorescent protein (GFP) foci on pCBG (infectious BaMV cDNA viral vector which can express GFP)‐inoculated Nicotiana benthamiana leaves at 5 days post‐inoculation, measured by fluorescence microscopy. Bar length, 1.0 mm. (B) Statistical analysis of the results obtained from (A); y ‐axis is the GFP focus size (mm 2 ). The numbers shown above the bars are the average and standard deviation of 24 and 35 foci from Luc ‐ and NbTRXh2 ‐knockdown plants, respectively. Asterisks indicate statistically significant differences of the indicated group analysed by Student's t ‐test (*** P
    Figure Legend Snippet: Cell‐to‐cell movement of Barley mosaic virus (BaMV) in Luc ‐ and NbTRXh2 ‐knockdown plants. (A) Areas of green fluorescent protein (GFP) foci on pCBG (infectious BaMV cDNA viral vector which can express GFP)‐inoculated Nicotiana benthamiana leaves at 5 days post‐inoculation, measured by fluorescence microscopy. Bar length, 1.0 mm. (B) Statistical analysis of the results obtained from (A); y ‐axis is the GFP focus size (mm 2 ). The numbers shown above the bars are the average and standard deviation of 24 and 35 foci from Luc ‐ and NbTRXh2 ‐knockdown plants, respectively. Asterisks indicate statistically significant differences of the indicated group analysed by Student's t ‐test (*** P

    Techniques Used: Plasmid Preparation, Fluorescence, Microscopy, Standard Deviation

    Co‐immunoprecipitation of Barley mosaic virus (BaMV) movement protein with NbTRXh2 and its derivatives. The green fluorescent protein (GFP)‐fused BaMV movement proteins (GFP‐TGBp1 and GFP‐TGBp2) were transiently expressed in Nicotiana benthamiana and subjected to immunoprecipitation by magnetic anti‐GFP beads. The pull‐down GFP or GFP‐fused proteins interacting with NbTRXh2 and its derivatives are indicated. The immunoprecipitated proteins and their expression levels (indicated as input) were analysed by Western blot analysis using anti‐His and anti‐GFP antibodies.
    Figure Legend Snippet: Co‐immunoprecipitation of Barley mosaic virus (BaMV) movement protein with NbTRXh2 and its derivatives. The green fluorescent protein (GFP)‐fused BaMV movement proteins (GFP‐TGBp1 and GFP‐TGBp2) were transiently expressed in Nicotiana benthamiana and subjected to immunoprecipitation by magnetic anti‐GFP beads. The pull‐down GFP or GFP‐fused proteins interacting with NbTRXh2 and its derivatives are indicated. The immunoprecipitated proteins and their expression levels (indicated as input) were analysed by Western blot analysis using anti‐His and anti‐GFP antibodies.

    Techniques Used: Immunoprecipitation, Expressing, Western Blot

    17) Product Images from "A Genetic Interaction Map of RNA Processing Factors Reveals Links Between Sem1/Dss1-Containing Complexes and mRNA Export and Splicing"

    Article Title: A Genetic Interaction Map of RNA Processing Factors Reveals Links Between Sem1/Dss1-Containing Complexes and mRNA Export and Splicing

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2008.11.012

    Sem1 is involved in mRNA export via the Sac3-Thp1 complex A) Scatter plot of correlation coefficients for each mutant compared to the profiles generated from SAC3 (y-axis) and THP1 (x-axis). B) Representative genetic interactions from the E-MAP that differentiate SEM1 , SAC3 , and THP1 from RPN10 and PRE9 . Blue and yellow indicate negative and positive genetic interactions, respectively. C) In situ hybridizations with a dT50 probe to detect accumulation of poly(A) RNA (top row). The bottom row is DAPI staining to detect nuclei. Cells were either kept at permissive temperature (30°C, left) or shifted to 16°C for 2 hours before fixation (right). D) Co-immunoprecipitations of Sem1 and Rpt6 with GFP-tagged proteins. The indicated strains were immunoprecipitated with a monoclonal GFP antibody, either with or without prior RNAse A treatment of the extract, and the blot was cut and probed with polyclonal antibodies against Sem1 or Rpt6. The right panel is 1/200th of the sample for the GFP IPs exposed identically to the left. E) Co-immunoprecipitations of Thp1-6HA with Sac3GFP in a sem1 Δ background. The immunoprecipitations were washed at room temperature with buffers containing the indicated amount of salt. The right panel represents1/20 th the sample for the GFP IPS. F) ). The left and right panels were exposed differently, and the Yra1 poly(A) blot was overexposed to allow visualization of the Yra1 band in the wild-type strain. G) Depiction of the export block identified by the poly(A) crosslinking in part F. Sac3-Thp1-Sem1 could facilitate an exchange of Sub2 for Mex67. See text for more details.
    Figure Legend Snippet: Sem1 is involved in mRNA export via the Sac3-Thp1 complex A) Scatter plot of correlation coefficients for each mutant compared to the profiles generated from SAC3 (y-axis) and THP1 (x-axis). B) Representative genetic interactions from the E-MAP that differentiate SEM1 , SAC3 , and THP1 from RPN10 and PRE9 . Blue and yellow indicate negative and positive genetic interactions, respectively. C) In situ hybridizations with a dT50 probe to detect accumulation of poly(A) RNA (top row). The bottom row is DAPI staining to detect nuclei. Cells were either kept at permissive temperature (30°C, left) or shifted to 16°C for 2 hours before fixation (right). D) Co-immunoprecipitations of Sem1 and Rpt6 with GFP-tagged proteins. The indicated strains were immunoprecipitated with a monoclonal GFP antibody, either with or without prior RNAse A treatment of the extract, and the blot was cut and probed with polyclonal antibodies against Sem1 or Rpt6. The right panel is 1/200th of the sample for the GFP IPs exposed identically to the left. E) Co-immunoprecipitations of Thp1-6HA with Sac3GFP in a sem1 Δ background. The immunoprecipitations were washed at room temperature with buffers containing the indicated amount of salt. The right panel represents1/20 th the sample for the GFP IPS. F) ). The left and right panels were exposed differently, and the Yra1 poly(A) blot was overexposed to allow visualization of the Yra1 band in the wild-type strain. G) Depiction of the export block identified by the poly(A) crosslinking in part F. Sac3-Thp1-Sem1 could facilitate an exchange of Sub2 for Mex67. See text for more details.

    Techniques Used: Mutagenesis, Generated, In Situ, Staining, Immunoprecipitation, Blocking Assay

    18) Product Images from "A conserved function for pericentromeric satellite DNA"

    Article Title: A conserved function for pericentromeric satellite DNA

    Journal: eLife

    doi: 10.7554/eLife.34122

    D1/HMGA1 loss-of-function results in micronuclei formation, and defective nuclear envelope integrity. ( A, B ) Control ( D1 LL03310 /+) ( A ) and D1 LL03310 / Df mutant ( B ) spermatogonial cells stained for DAPI (red), Vasa (blue) and LaminDm 0 (green). Arrow indicates micronucleus. Bars: 5 μm. ( C ) Quantification of micronuclei-containing cells from +/+ control (n = 269) and D1 LL03310 /Df (n = 334) from three independent experiments. p-Value from student’s t-test is shown. Error bars: SD. ( D, E ) siControl ( D ) and siHMGA1 transfected ( E ) MOVAS cells stained for DAPI (blue), HMGA1 (red) and Lamin (green). Arrow indicates micronucleus. ( F ) Quantification of micronuclei-containing cells in siControl (n = 518) and siHMGA1 (n = 588) transfected cells from four independent experiments. ( G, H ) Control ( D1 LL03310 /+) ( G ) and D1 LL03310 / Df ( H ) spermatogonia expressing nls-GFP (green) stained for Vasa (blue) and LaminDm 0 (red). nlsGFP was observed in cytoplasm in D1 LL03310 / Df spermatogonia. ( I ) Quantification of spermatogonia with cytoplasmic GFP ( > 1 μm exclusions or pan-cytoplasmic) in D1 LL03310 /+ (n = 810) and D1 LL03310 / Df (n = 780) testes from two independent experiments. ( J, K ) D1 LL03310 /+ ( J ) and D1 LL03310 / Df ( K ) spermatogonia expressing ER-GFP marker (green) stained for Vasa (blue) and LaminDm 0 (red). Arrowhead points to ER marker-positive micronucleus. Arrows point to site of weak nuclear LaminDm 0 staining. ( L, M ) Control ( D1 LL03310 /+) ( L ) and D1 LL03310 / Df ( M ) spermatogonia stained for Vasa (blue) and LaminDm 0 (green) and Otefin (red). Arrowhead points to Otefin-containing micronucleus. Arrows point to site of weak nuclear LaminDm 0 staining.
    Figure Legend Snippet: D1/HMGA1 loss-of-function results in micronuclei formation, and defective nuclear envelope integrity. ( A, B ) Control ( D1 LL03310 /+) ( A ) and D1 LL03310 / Df mutant ( B ) spermatogonial cells stained for DAPI (red), Vasa (blue) and LaminDm 0 (green). Arrow indicates micronucleus. Bars: 5 μm. ( C ) Quantification of micronuclei-containing cells from +/+ control (n = 269) and D1 LL03310 /Df (n = 334) from three independent experiments. p-Value from student’s t-test is shown. Error bars: SD. ( D, E ) siControl ( D ) and siHMGA1 transfected ( E ) MOVAS cells stained for DAPI (blue), HMGA1 (red) and Lamin (green). Arrow indicates micronucleus. ( F ) Quantification of micronuclei-containing cells in siControl (n = 518) and siHMGA1 (n = 588) transfected cells from four independent experiments. ( G, H ) Control ( D1 LL03310 /+) ( G ) and D1 LL03310 / Df ( H ) spermatogonia expressing nls-GFP (green) stained for Vasa (blue) and LaminDm 0 (red). nlsGFP was observed in cytoplasm in D1 LL03310 / Df spermatogonia. ( I ) Quantification of spermatogonia with cytoplasmic GFP ( > 1 μm exclusions or pan-cytoplasmic) in D1 LL03310 /+ (n = 810) and D1 LL03310 / Df (n = 780) testes from two independent experiments. ( J, K ) D1 LL03310 /+ ( J ) and D1 LL03310 / Df ( K ) spermatogonia expressing ER-GFP marker (green) stained for Vasa (blue) and LaminDm 0 (red). Arrowhead points to ER marker-positive micronucleus. Arrows point to site of weak nuclear LaminDm 0 staining. ( L, M ) Control ( D1 LL03310 /+) ( L ) and D1 LL03310 / Df ( M ) spermatogonia stained for Vasa (blue) and LaminDm 0 (green) and Otefin (red). Arrowhead points to Otefin-containing micronucleus. Arrows point to site of weak nuclear LaminDm 0 staining.

    Techniques Used: Mutagenesis, Staining, Transfection, Expressing, Marker

    D1 bundles satellite DNA from heterologous chromosomes to form chromocenter. ( A, B ) C2C12 cells expressing GFP only (blue) ( A ) or GFP-D1 (blue) ( B ) stained for DAPI (red). Dotted lines indicate nucleus. ( C ) Quantification of chromocenter number relative to expression level of GFP (n = 29) or GFP-D1 (n = 47). P value and R 2 value are indicated from linear regression analysis. ( D ) FISH against LacO (red) and {AATAT} n (green) on mitotic neuroblast chromosomes from the LacO strain stained for DAPI (blue), indicating the sites of LacO insertion (arrows). ( E, F ) FISH against LacO (red) and {AATAT} n (green) in spermatogonia expressing GFP-D1 (blue) ( E ) or GFP-LacI-D1 (blue) ( F ). Arrows indicate location of LacO sequence. ( G ) AATAT-LacO distance (nm) in GFP-D1 (n = 97) and GFP-LacI-D1 (n = 69) expressing spermatogonia. P value from student’s t-test is shown. Error bars: SD. All scale bars: 5 μm.
    Figure Legend Snippet: D1 bundles satellite DNA from heterologous chromosomes to form chromocenter. ( A, B ) C2C12 cells expressing GFP only (blue) ( A ) or GFP-D1 (blue) ( B ) stained for DAPI (red). Dotted lines indicate nucleus. ( C ) Quantification of chromocenter number relative to expression level of GFP (n = 29) or GFP-D1 (n = 47). P value and R 2 value are indicated from linear regression analysis. ( D ) FISH against LacO (red) and {AATAT} n (green) on mitotic neuroblast chromosomes from the LacO strain stained for DAPI (blue), indicating the sites of LacO insertion (arrows). ( E, F ) FISH against LacO (red) and {AATAT} n (green) in spermatogonia expressing GFP-D1 (blue) ( E ) or GFP-LacI-D1 (blue) ( F ). Arrows indicate location of LacO sequence. ( G ) AATAT-LacO distance (nm) in GFP-D1 (n = 97) and GFP-LacI-D1 (n = 69) expressing spermatogonia. P value from student’s t-test is shown. Error bars: SD. All scale bars: 5 μm.

    Techniques Used: Expressing, Staining, Fluorescence In Situ Hybridization, Sequencing

    D1/HMGA1 loss of function results in micronuclei formation due to nuclear budding during interphase. ( A, B ) Time-lapse live imaging of control (+/+) ( A ) and D1 LL03310 /Df ( B ) spermatogonial cells expressing Df31-GFP as a nuclear marker and H2Av-RFP as a DNA marker. ( C, D ) Time-lapse live imaging of siControl ( C ) and siHMGA1 ( D ) MOVAS cells stained with Hoechst 33342. Arrowheads indicate site of micronucleus budding. Time is indicated in mm:ss. Scale bars: 5 μm.
    Figure Legend Snippet: D1/HMGA1 loss of function results in micronuclei formation due to nuclear budding during interphase. ( A, B ) Time-lapse live imaging of control (+/+) ( A ) and D1 LL03310 /Df ( B ) spermatogonial cells expressing Df31-GFP as a nuclear marker and H2Av-RFP as a DNA marker. ( C, D ) Time-lapse live imaging of siControl ( C ) and siHMGA1 ( D ) MOVAS cells stained with Hoechst 33342. Arrowheads indicate site of micronucleus budding. Time is indicated in mm:ss. Scale bars: 5 μm.

    Techniques Used: Imaging, Expressing, Marker, Staining

    Multi-AT-hook proteins, D1 and HMGA1, are required for chromocenter formation in Drosophila and mouse cells. ( A ) Schematic of pericentromeric heterochromatin being organized into the chromocenter. ( B ) FISH against {AATAT} n satellite (red) on the Drosophila neuroblast mitotic chromosomes co-stained with DAPI (blue) indicating the location of {AATAT} n in the Drosophila genome. ( C ) FISH against {AATAT} n satellite (red) in spermatogonial cells immunostained for H3K9me2 (blue) and D1 (green). Dotted lines indicate nucleus. Bars: 5 µm. ( D ) Drosophila neuroblast mitotic chromosomes stained for D1 (green), phospho-histone H3 Serine 10 (pH3-S10) (blue) and Cid/CENP-A (red). ( E–G ) FISH against the mouse major satellite (green) on C2C12 mitotic chromosomes co-stained with DAPI (blue) ( E ), in interphase MOVAS cells co-stained for DAPI (blue) and HMGA1 (red) ( F ) and in MOVAS cells expressing GFP-D1 (blue) stained for HMGA1 (red) ( G ). ( H, I ) FISH against {AATAT} n satellite (red) in control ( D1 LL03310 /+) ( H ) and D1 LL03310 / Df ( I ) spermatogonial cells stained for DAPI (blue) and Vasa (green). ( J ) Quantification of spermatogonial cells with disrupted chromocenters (+/+ control n = 117, D1 LL03310 / Df n = 89) from three independent experiments. p-Value from student’s t-test is shown. Error bars: SD. ( K, L ) FISH against the major satellite (green) in siControl ( K ) and siHMGA1 ( L ) transfected MOVAS cells co-stained with DAPI (blue). ( M ) Quantification of cells with disrupted chromocenters from siControl (n = 304) and siHMGA1 (n = 329) from three independent experiments.
    Figure Legend Snippet: Multi-AT-hook proteins, D1 and HMGA1, are required for chromocenter formation in Drosophila and mouse cells. ( A ) Schematic of pericentromeric heterochromatin being organized into the chromocenter. ( B ) FISH against {AATAT} n satellite (red) on the Drosophila neuroblast mitotic chromosomes co-stained with DAPI (blue) indicating the location of {AATAT} n in the Drosophila genome. ( C ) FISH against {AATAT} n satellite (red) in spermatogonial cells immunostained for H3K9me2 (blue) and D1 (green). Dotted lines indicate nucleus. Bars: 5 µm. ( D ) Drosophila neuroblast mitotic chromosomes stained for D1 (green), phospho-histone H3 Serine 10 (pH3-S10) (blue) and Cid/CENP-A (red). ( E–G ) FISH against the mouse major satellite (green) on C2C12 mitotic chromosomes co-stained with DAPI (blue) ( E ), in interphase MOVAS cells co-stained for DAPI (blue) and HMGA1 (red) ( F ) and in MOVAS cells expressing GFP-D1 (blue) stained for HMGA1 (red) ( G ). ( H, I ) FISH against {AATAT} n satellite (red) in control ( D1 LL03310 /+) ( H ) and D1 LL03310 / Df ( I ) spermatogonial cells stained for DAPI (blue) and Vasa (green). ( J ) Quantification of spermatogonial cells with disrupted chromocenters (+/+ control n = 117, D1 LL03310 / Df n = 89) from three independent experiments. p-Value from student’s t-test is shown. Error bars: SD. ( K, L ) FISH against the major satellite (green) in siControl ( K ) and siHMGA1 ( L ) transfected MOVAS cells co-stained with DAPI (blue). ( M ) Quantification of cells with disrupted chromocenters from siControl (n = 304) and siHMGA1 (n = 329) from three independent experiments.

    Techniques Used: Fluorescence In Situ Hybridization, Staining, Expressing, Transfection

    19) Product Images from "Iron Supply via NCOA4-Mediated Ferritin Degradation Maintains Mitochondrial Functions"

    Article Title: Iron Supply via NCOA4-Mediated Ferritin Degradation Maintains Mitochondrial Functions

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00010-19

    The ferritin light chain is required for iron supply to mitochondria under iron-deprived conditions. (A) Wild-type (WT) and FTL knockout (KO) HeLa cells treated with 100 μM DFO for the indicated times were immunoblotted with the indicated antibodies. (B) The PNS was obtained from WT and FTL KO HeLa cells treated with 100 μM DFO for 24 h. Shown are results from BN-PAGE followed by immunoblotting with the indicated antibodies. (C) Representative images of WT and FTL KO cells incubated with 100 μM DFO for 24 h and immunostained with anti-LAMP2 and anti-FTH1 antibodies. Bar = 10 μm. (D) WT and FTL KO HeLa cells expressing GFP-NCOA4 incubated with 100 μM DFO for 24 h. Immunostaining with anti-FTH1 antibodies was performed. Arrows indicate GFP-NCOA4 puncta that colocalized with FTH1. Bars = 10 μm. (E) FTL WT and KO cells expressing GFP-NCOA4 or GFP were incubated for 48 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-GFP magnetic beads. Immunoprecipitates were analyzed by Western blotting. (F) OCRs in WT and FTL KO cells were measured. OCRs were obtained from four independent wells. (G) Results of quantification of basal respiration, ATP production, and maximum respiration. The results are from five independent wells. (H) Mitochondria were isolated from FTL WT and KO cells expressing FLAG-FTL or an empty vector incubated with 100 μM DFO for 6 h. BN-PAGE and SDS-PAGE followed by immunoblotting were performed with the indicated antibodies. (I) FTL KO cells, FLAG-FTL cells, or empty vector cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (J and K) FTL KO cells were transfected with FLAG-FTL or an empty vector with (J) or without (K) GFP-NCOA4. Cells treated with 100 μM DFO for 24 h were immunostained with anti-LAMP2 and anti-FTH1 (K) or anti-FTH1 (J) antibodies. Bars = 10 μm. (L) OCRs in FTL KO cells transfected with FLAG-FTL or an empty vector were measured. OCRs were obtained from five independent wells. (M) Results of quantification of basal respiration, ATP production, and maximum respiration from panel L. Error bars represent mean values ± SEM. ns, not significant; *, P  
    Figure Legend Snippet: The ferritin light chain is required for iron supply to mitochondria under iron-deprived conditions. (A) Wild-type (WT) and FTL knockout (KO) HeLa cells treated with 100 μM DFO for the indicated times were immunoblotted with the indicated antibodies. (B) The PNS was obtained from WT and FTL KO HeLa cells treated with 100 μM DFO for 24 h. Shown are results from BN-PAGE followed by immunoblotting with the indicated antibodies. (C) Representative images of WT and FTL KO cells incubated with 100 μM DFO for 24 h and immunostained with anti-LAMP2 and anti-FTH1 antibodies. Bar = 10 μm. (D) WT and FTL KO HeLa cells expressing GFP-NCOA4 incubated with 100 μM DFO for 24 h. Immunostaining with anti-FTH1 antibodies was performed. Arrows indicate GFP-NCOA4 puncta that colocalized with FTH1. Bars = 10 μm. (E) FTL WT and KO cells expressing GFP-NCOA4 or GFP were incubated for 48 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-GFP magnetic beads. Immunoprecipitates were analyzed by Western blotting. (F) OCRs in WT and FTL KO cells were measured. OCRs were obtained from four independent wells. (G) Results of quantification of basal respiration, ATP production, and maximum respiration. The results are from five independent wells. (H) Mitochondria were isolated from FTL WT and KO cells expressing FLAG-FTL or an empty vector incubated with 100 μM DFO for 6 h. BN-PAGE and SDS-PAGE followed by immunoblotting were performed with the indicated antibodies. (I) FTL KO cells, FLAG-FTL cells, or empty vector cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (J and K) FTL KO cells were transfected with FLAG-FTL or an empty vector with (J) or without (K) GFP-NCOA4. Cells treated with 100 μM DFO for 24 h were immunostained with anti-LAMP2 and anti-FTH1 (K) or anti-FTH1 (J) antibodies. Bars = 10 μm. (L) OCRs in FTL KO cells transfected with FLAG-FTL or an empty vector were measured. OCRs were obtained from five independent wells. (M) Results of quantification of basal respiration, ATP production, and maximum respiration from panel L. Error bars represent mean values ± SEM. ns, not significant; *, P  

    Techniques Used: Knock-Out, Polyacrylamide Gel Electrophoresis, Incubation, Expressing, Immunostaining, Immunoprecipitation, Magnetic Beads, Western Blot, Isolation, Plasmid Preparation, SDS Page, Transfection

    20) Product Images from "Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions *Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions * ♦"

    Article Title: Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions *Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.440396

    VCA domain of N-WASP co-localizes with p120-catenin and induces cortical actin formation. A–C , HPAEC transfected with the GFP-VCA or GFP-CA mutants were stimulated with 50 n m thrombin for 30 min, after which cells were fixed and stained with rhodamine-phalloidin
    Figure Legend Snippet: VCA domain of N-WASP co-localizes with p120-catenin and induces cortical actin formation. A–C , HPAEC transfected with the GFP-VCA or GFP-CA mutants were stimulated with 50 n m thrombin for 30 min, after which cells were fixed and stained with rhodamine-phalloidin

    Techniques Used: Transfection, Staining

    VCA domain of N-WASP interacts with p120-catenin. Left , COS7 cells were transfected with the indicated constructs. Forty-eight h later, the lysates were immunoprecipitated with anti-GFP antibodies followed by immunoblotting with the indicated antibodies
    Figure Legend Snippet: VCA domain of N-WASP interacts with p120-catenin. Left , COS7 cells were transfected with the indicated constructs. Forty-eight h later, the lysates were immunoprecipitated with anti-GFP antibodies followed by immunoblotting with the indicated antibodies

    Techniques Used: Transfection, Construct, Immunoprecipitation

    21) Product Images from "Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos"

    Article Title: Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos

    Journal: Physiology and Molecular Biology of Plants

    doi: 10.1007/s12298-018-00641-5

    Schematic diagram of transformation vectors pXQD12 and pXQD70. LB, T-DNA left border; RB, T-DNA right border; 35S, CaMV 35S promoter; ZmUbi1, maize Ubiquitin 1 promoter; sGFP , a synthetic green fluorescence protein; HPT , hygromycin phosphotransferase gene under the control of 2X CaMV 35S promoter; NPTII , neomycin phosphotransferase gene under the control of 2X CaMV 35S promoter. The backbone of pXQD12 and pXQD70 derive from pCAMBIA1300 and pPZP211, respectively
    Figure Legend Snippet: Schematic diagram of transformation vectors pXQD12 and pXQD70. LB, T-DNA left border; RB, T-DNA right border; 35S, CaMV 35S promoter; ZmUbi1, maize Ubiquitin 1 promoter; sGFP , a synthetic green fluorescence protein; HPT , hygromycin phosphotransferase gene under the control of 2X CaMV 35S promoter; NPTII , neomycin phosphotransferase gene under the control of 2X CaMV 35S promoter. The backbone of pXQD12 and pXQD70 derive from pCAMBIA1300 and pPZP211, respectively

    Techniques Used: Transformation Assay, Fluorescence

    Effect of different co-culture methods on the efficiency of Agrobacterium -mediated transformation of maize immature embryos (IEs). a Transient expression of GFP in Agrobacterium -infected maize calli (8 d after inoculation, 3 d for co-culture and 5 d for restoration; left, white light; right, GFP3 filter; bar = 0.6 mm). b – g Comparison of the two co-culture methods ( MC medium; DC dry filter paper) under different conditions in terms of percentage of GFP positive calli ( b plasmids pXQD12 and pXQD70 in Agrobacterium strain AGL1; c Agrobacterium strains AGL1 and EHA105 carrying pXQD12; d – g inoculation durations of 5 min, 10 min, 15 min, 20 min and 25 min of AGL1/pXQD12, AGL1/pXQD70, EHA105/pXQD12 or EHA105/pXQD70. Data in b – g are shown as mean ± S.D. Student’s t -tests were used to generate the p values: t test: * p
    Figure Legend Snippet: Effect of different co-culture methods on the efficiency of Agrobacterium -mediated transformation of maize immature embryos (IEs). a Transient expression of GFP in Agrobacterium -infected maize calli (8 d after inoculation, 3 d for co-culture and 5 d for restoration; left, white light; right, GFP3 filter; bar = 0.6 mm). b – g Comparison of the two co-culture methods ( MC medium; DC dry filter paper) under different conditions in terms of percentage of GFP positive calli ( b plasmids pXQD12 and pXQD70 in Agrobacterium strain AGL1; c Agrobacterium strains AGL1 and EHA105 carrying pXQD12; d – g inoculation durations of 5 min, 10 min, 15 min, 20 min and 25 min of AGL1/pXQD12, AGL1/pXQD70, EHA105/pXQD12 or EHA105/pXQD70. Data in b – g are shown as mean ± S.D. Student’s t -tests were used to generate the p values: t test: * p

    Techniques Used: Co-Culture Assay, Transformation Assay, Expressing, Infection

    22) Product Images from "Transcriptional Inhibition of Sp-IAG by Crustacean Female Sex Hormone in the Mud Crab, Scylla paramamosain"

    Article Title: Transcriptional Inhibition of Sp-IAG by Crustacean Female Sex Hormone in the Mud Crab, Scylla paramamosain

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21155300

    Effect of Sp-STAT dsRNA on Sp-IAG expression in vitro. Expression of Sp-STAT analyzed at hour 2 and 4 ( A ). The expression of Sp-IAG was analyzed after incubated for 4 h, water and GFP (green fluorescent protein gene) were used as blank and negative controls, respectively ( B ). Data presented as mean ± SEM ( n = 4). Different letters indicate statistical significance at p
    Figure Legend Snippet: Effect of Sp-STAT dsRNA on Sp-IAG expression in vitro. Expression of Sp-STAT analyzed at hour 2 and 4 ( A ). The expression of Sp-IAG was analyzed after incubated for 4 h, water and GFP (green fluorescent protein gene) were used as blank and negative controls, respectively ( B ). Data presented as mean ± SEM ( n = 4). Different letters indicate statistical significance at p

    Techniques Used: Expressing, In Vitro, Incubation

    23) Product Images from "Exome and whole genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity"

    Article Title: Exome and whole genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity

    Journal: Nature genetics

    doi: 10.1038/ng.2591

    Recurrent somatic alterations in ELMO1 , DOCK2, and other RAC1 Guanine Nucleotide Exchange Factors (GEFs) a) Schematic of protein alterations in DOCK2 and ELMO1 detected by WES. Coding alterations in EAC are colored either black (missense) or red (splice site/nonsense); silent mutations are depicted in gray. Conserved domain mapping is from UniProt; SH3, SRC Homology 3; DHR, Dlg homologous region, ELMO, Engulfment and Cell Motility; PH, Pleckstrin homology. b) Sample frequency (left) of candidate ELMO1 and DOCK2 as well as other Rac1-activating guanine nucleotide exchange factors in 145 WES EACs. c) ELMO1 wild-type or mutants (or GFP control) were expressed in NIH/3T3 cells using retroviral transduction with the pBabe vector. Cells were plated in matrigel invasion chambers with full serum containing medium in the lower chamber only, and invading cells from four fields were counted. Invading cells of 3 independent replicates are shown. Error bars represent S.D. P -values compare mutant ELMO1 to wild-type. n.s., not significant. Student’s t-test.
    Figure Legend Snippet: Recurrent somatic alterations in ELMO1 , DOCK2, and other RAC1 Guanine Nucleotide Exchange Factors (GEFs) a) Schematic of protein alterations in DOCK2 and ELMO1 detected by WES. Coding alterations in EAC are colored either black (missense) or red (splice site/nonsense); silent mutations are depicted in gray. Conserved domain mapping is from UniProt; SH3, SRC Homology 3; DHR, Dlg homologous region, ELMO, Engulfment and Cell Motility; PH, Pleckstrin homology. b) Sample frequency (left) of candidate ELMO1 and DOCK2 as well as other Rac1-activating guanine nucleotide exchange factors in 145 WES EACs. c) ELMO1 wild-type or mutants (or GFP control) were expressed in NIH/3T3 cells using retroviral transduction with the pBabe vector. Cells were plated in matrigel invasion chambers with full serum containing medium in the lower chamber only, and invading cells from four fields were counted. Invading cells of 3 independent replicates are shown. Error bars represent S.D. P -values compare mutant ELMO1 to wild-type. n.s., not significant. Student’s t-test.

    Techniques Used: Transduction, Plasmid Preparation, Mutagenesis

    24) Product Images from "Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway"

    Article Title: Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002369

    Cellular localization of T. cruzi enzymes of the GPI biosynthetic pathway. Epimastigotes were transiently transfected with the plasmids pTREX-TcDPM1-GFP ( A ), pTREX-TcGPI3-GFP ( B ), pTREX-TcGPI12-GFP ( C ) or pTREXnGFP as a control plasmid ( D ) and ( E ). Transfected parasites were fixed with 4% paraformaldehyde, incubated with the ER marker anti-BiP (1∶1000) and the secondary antibody conjugated to Alexa 555 (1∶1000). Cells were also stained with DAPI showing the nuclear and kinetoplast DNA. In panel E , parasites that were not incubated with the primary, anti-BiP antibody are shown as negative controls. Images were captured with the Nikon Eclipse Ti fluorescence microscope. Scale bars: 5 µm.
    Figure Legend Snippet: Cellular localization of T. cruzi enzymes of the GPI biosynthetic pathway. Epimastigotes were transiently transfected with the plasmids pTREX-TcDPM1-GFP ( A ), pTREX-TcGPI3-GFP ( B ), pTREX-TcGPI12-GFP ( C ) or pTREXnGFP as a control plasmid ( D ) and ( E ). Transfected parasites were fixed with 4% paraformaldehyde, incubated with the ER marker anti-BiP (1∶1000) and the secondary antibody conjugated to Alexa 555 (1∶1000). Cells were also stained with DAPI showing the nuclear and kinetoplast DNA. In panel E , parasites that were not incubated with the primary, anti-BiP antibody are shown as negative controls. Images were captured with the Nikon Eclipse Ti fluorescence microscope. Scale bars: 5 µm.

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Marker, Staining, Fluorescence, Microscopy

    25) Product Images from "Calcium dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel"

    Article Title: Calcium dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel

    Journal: Nature cell biology

    doi: 10.1038/ncb2887

    P2XA suppresses Rab11a activity A. Immunoprecipitation of GFP or P2XA-GFP from wild-type cells. P2XA is indicated with a closed arrow, Rab11a is indicated with an open arrow. B. Immunoprecipitation of Rab11a-RFP or RFP from P2XA-GFP co-expressing cells. Lysates were incubated with anti-GFP beads and input, bound and not bound fractions probed with either anti-GFP or anti-RFP antibodies. C. Localization of P2XA-RFP and Rab11a-GFP in wild-type cells. P2XA and Rab11a co-localize on the CV. D. Overexpression of Rab11a-RFP results in osmoregulation defects similar to those observed for P2XA − cells. Quantification of fluorescence levels in three clones (clone 1 = 1052 A.U., clone 2 = 2183 A.U., clone 3 = 4911 A.U.) shows that higher expression results in more small vacuoles that fail to fuse (top panel) and less recovery from osmotic shock (bottom panel). E. Quantification of Rab11a-GTP levels in the three Rab11a-RFP expressing clones by immunoprecipitation with a Rab11a-GTP specific antibody as described in Materials and Methods (I = input, B = bound, NB = not bound). A paired T test revealed that the ratio of GTP and GDP bound Rab11a was not significantly different between the three clones (p=0.89 for each pair). Error bars represent s.e.m. from n=3 three independent experiments. Statistical source data for Fig 5E can be found in Supplementary Table 2 .F. Immunoprecipitation of Rab11a-GTP from AX4 cells expressing Rab11a-RFP, P2XA − cells expressing Rab11a-RFP, wild type cells expressing dominant negative Rab11a 22 fused to RFP, and wild type cells expressing constitutively active Rab11a 22 fused to RFP. The level of Rab11a-RFP expression (input) is comparable between all strains ( > 1.25 fold difference between highest and lowest). G. Quantification of GTP and GDP bound Rab11a . A paired T test revealed a significant increase (2.2 fold) in the levels of GTP bound Rab11a in P2XA − cells compared to wild type cells (p
    Figure Legend Snippet: P2XA suppresses Rab11a activity A. Immunoprecipitation of GFP or P2XA-GFP from wild-type cells. P2XA is indicated with a closed arrow, Rab11a is indicated with an open arrow. B. Immunoprecipitation of Rab11a-RFP or RFP from P2XA-GFP co-expressing cells. Lysates were incubated with anti-GFP beads and input, bound and not bound fractions probed with either anti-GFP or anti-RFP antibodies. C. Localization of P2XA-RFP and Rab11a-GFP in wild-type cells. P2XA and Rab11a co-localize on the CV. D. Overexpression of Rab11a-RFP results in osmoregulation defects similar to those observed for P2XA − cells. Quantification of fluorescence levels in three clones (clone 1 = 1052 A.U., clone 2 = 2183 A.U., clone 3 = 4911 A.U.) shows that higher expression results in more small vacuoles that fail to fuse (top panel) and less recovery from osmotic shock (bottom panel). E. Quantification of Rab11a-GTP levels in the three Rab11a-RFP expressing clones by immunoprecipitation with a Rab11a-GTP specific antibody as described in Materials and Methods (I = input, B = bound, NB = not bound). A paired T test revealed that the ratio of GTP and GDP bound Rab11a was not significantly different between the three clones (p=0.89 for each pair). Error bars represent s.e.m. from n=3 three independent experiments. Statistical source data for Fig 5E can be found in Supplementary Table 2 .F. Immunoprecipitation of Rab11a-GTP from AX4 cells expressing Rab11a-RFP, P2XA − cells expressing Rab11a-RFP, wild type cells expressing dominant negative Rab11a 22 fused to RFP, and wild type cells expressing constitutively active Rab11a 22 fused to RFP. The level of Rab11a-RFP expression (input) is comparable between all strains ( > 1.25 fold difference between highest and lowest). G. Quantification of GTP and GDP bound Rab11a . A paired T test revealed a significant increase (2.2 fold) in the levels of GTP bound Rab11a in P2XA − cells compared to wild type cells (p

    Techniques Used: Activity Assay, Immunoprecipitation, Expressing, Incubation, Over Expression, Fluorescence, Clone Assay, Dominant Negative Mutation

    cnrF is a cytosolic calcium dependent Rab11a GAP that interacts with P2XA and Rab11a A. Rescue of cnrF mutant null phenotype by full-length (1-670) cnrF-RFP and a truncated (1-410) form of cnrF-RFP that lacks the EF hands. Cell lines were selected based on RFP expression levels. Both CnrF − CnrF 1-670 Low (2064 A.U.), CnrFCnrF 1- 670 High (10106 A.U.) and CnrF − CnrF 1-410 High (9987 A.U.) cells show an indistinguishable hypoosmotic shock response to wild type (Paired T test p > 0.01). However, CnrF − CnrF 1-410 Low (2111 A.U.) cells fail to rescue the mutant phenotype (Paired T test p > 0.05). Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 100 cells. Statistical source data for Fig 8A can be found in Supplementary Table 2 .B. Rescue of cnrF − mutant phenotype by a point mutated forms of cnrF-RFP that disrupt the EF hands (cnrF(E623Q/D659Q)) or rab GAP activity (cnrF(R270A)). CnrF( E623Q/D659Q )-RFP low cells (2044 a.u.) fail to rescue the mutant phenotype (Paired T test p > 0.05), whereas cells expressing high levels of CnrF( E623Q/D659Q )-RFP (9946 A.U.) show a similar hypoosmotic shock response to wild type (Paired T test p > 0.05). Both low levels of CnrF (R270 )-RFP expression (1998 A.U.) and high levels of CnrF (R270 )-RFP expression (10042 A.U.) fail to rescue the mutant phenotype (Paired T test p > 0.05). Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 100 cells. Statistical source data for Fig 8B can be found in Supplementary Table 2 . C. CnrF:GFP expression in wild type cells. Scale bar = 5 μm. D. Rab11a and P2XA (but not Rab8a) bind GST-CnrF 1-670 or GST-CnrF 1-410 , but not GST-RabGAP421 control (a related Dictyostelium EF hand domain containing RabGAP (DDB_G0275421)). E. Rab11a binding to CnrF is not calcium dependent. Varying amounts of protein from cells expressing Rab11a:RFP were incubated with beads bound with GST-CnrF 1-670 or GST-CnrF 1-410 in 20mM Ca 2+ or 100mM EGTA. F. Proposed model of P2XA regulated vesicle fusion. Maturing vacuoles expresses P2XA, Rab11a-GTP and Drainin. Once tethered, P2XA undergoes a ‘ring to patch’ transition so that P2XA is only expressed at the plasma membrane contact site. Concentration of P2XA activity leads to a localized increase in calcium ions as they pass through the active channel. Consequently, P2XA bound CnrF is activated as Ca 2+ binds to the EF hand domain on CnrF, leading to the hydrolysis and inactivation of Rab11a-GTP, and therefore inactivation of Drainin at the plasma membrane contact site.
    Figure Legend Snippet: cnrF is a cytosolic calcium dependent Rab11a GAP that interacts with P2XA and Rab11a A. Rescue of cnrF mutant null phenotype by full-length (1-670) cnrF-RFP and a truncated (1-410) form of cnrF-RFP that lacks the EF hands. Cell lines were selected based on RFP expression levels. Both CnrF − CnrF 1-670 Low (2064 A.U.), CnrFCnrF 1- 670 High (10106 A.U.) and CnrF − CnrF 1-410 High (9987 A.U.) cells show an indistinguishable hypoosmotic shock response to wild type (Paired T test p > 0.01). However, CnrF − CnrF 1-410 Low (2111 A.U.) cells fail to rescue the mutant phenotype (Paired T test p > 0.05). Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 100 cells. Statistical source data for Fig 8A can be found in Supplementary Table 2 .B. Rescue of cnrF − mutant phenotype by a point mutated forms of cnrF-RFP that disrupt the EF hands (cnrF(E623Q/D659Q)) or rab GAP activity (cnrF(R270A)). CnrF( E623Q/D659Q )-RFP low cells (2044 a.u.) fail to rescue the mutant phenotype (Paired T test p > 0.05), whereas cells expressing high levels of CnrF( E623Q/D659Q )-RFP (9946 A.U.) show a similar hypoosmotic shock response to wild type (Paired T test p > 0.05). Both low levels of CnrF (R270 )-RFP expression (1998 A.U.) and high levels of CnrF (R270 )-RFP expression (10042 A.U.) fail to rescue the mutant phenotype (Paired T test p > 0.05). Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 100 cells. Statistical source data for Fig 8B can be found in Supplementary Table 2 . C. CnrF:GFP expression in wild type cells. Scale bar = 5 μm. D. Rab11a and P2XA (but not Rab8a) bind GST-CnrF 1-670 or GST-CnrF 1-410 , but not GST-RabGAP421 control (a related Dictyostelium EF hand domain containing RabGAP (DDB_G0275421)). E. Rab11a binding to CnrF is not calcium dependent. Varying amounts of protein from cells expressing Rab11a:RFP were incubated with beads bound with GST-CnrF 1-670 or GST-CnrF 1-410 in 20mM Ca 2+ or 100mM EGTA. F. Proposed model of P2XA regulated vesicle fusion. Maturing vacuoles expresses P2XA, Rab11a-GTP and Drainin. Once tethered, P2XA undergoes a ‘ring to patch’ transition so that P2XA is only expressed at the plasma membrane contact site. Concentration of P2XA activity leads to a localized increase in calcium ions as they pass through the active channel. Consequently, P2XA bound CnrF is activated as Ca 2+ binds to the EF hand domain on CnrF, leading to the hydrolysis and inactivation of Rab11a-GTP, and therefore inactivation of Drainin at the plasma membrane contact site.

    Techniques Used: Mutagenesis, Expressing, Activity Assay, Binding Assay, Incubation, Concentration Assay

    P2XA − cells exhibit aberrant contractile vacuole number, dynamics and morphology A. Fluorescence images of wild-type and P2XA − cells expressing Dajumin-GFP during CV cycling. Closed arrows indicate points in the CV cycle where a CV fuses to the plasma membrane and water is expelled. Numbers represent the time in seconds after changing the media from KK2 to water. Scale bar = 5μm. B. P2XA − cells have more vacuoles per cell than wild-type. P2XA − cells overexpressing P2XA:GFP have slightly more vacuoles per cell than wild-type, but fewer than P2XA − cells. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 cells. Statistical source data for Fig 2B can be found in Supplementary Table 2 .C. Fluorescence images of wild-type and P2XA − cell expressing Dajumin-GFP taken within the plane of the middle of the cell. Numbers represent the time in seconds after changing the media from KK2 to water. Scale bar = 5μm. D. The vacuoles in P2XA -cells undergo far fewer fusion/expulsion events than wild-type vacuoles during the time course. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 cells. Statistical source data for Fig 2D can be found in Supplementary Table 2 . E. The time taken for a CV to complete a cycle was twice as long for a P2XA − vacuole compared to wild-type vacuoles. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 vacuoles. Statistical source data for Fig 2E can be found in Supplementary Table 2 .F. P2XA -cells have larger numbers of small vacuoles than wild-type cells, and very few bigger vacuoles. P2XA − cells overexpressing P2XA:GFP have more small vacuoles than wild-type cells, but the number of bigger vacuoles is similar to wild-type cells. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 cells. Statistical source data for Fig 2F can be found in Supplementary Table 2 .G. Zoom of fluorescence images of P2XA − cell from panel A expressing Dajumin-GFP. White arrows indicate a “budding” vacuole.
    Figure Legend Snippet: P2XA − cells exhibit aberrant contractile vacuole number, dynamics and morphology A. Fluorescence images of wild-type and P2XA − cells expressing Dajumin-GFP during CV cycling. Closed arrows indicate points in the CV cycle where a CV fuses to the plasma membrane and water is expelled. Numbers represent the time in seconds after changing the media from KK2 to water. Scale bar = 5μm. B. P2XA − cells have more vacuoles per cell than wild-type. P2XA − cells overexpressing P2XA:GFP have slightly more vacuoles per cell than wild-type, but fewer than P2XA − cells. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 cells. Statistical source data for Fig 2B can be found in Supplementary Table 2 .C. Fluorescence images of wild-type and P2XA − cell expressing Dajumin-GFP taken within the plane of the middle of the cell. Numbers represent the time in seconds after changing the media from KK2 to water. Scale bar = 5μm. D. The vacuoles in P2XA -cells undergo far fewer fusion/expulsion events than wild-type vacuoles during the time course. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 cells. Statistical source data for Fig 2D can be found in Supplementary Table 2 . E. The time taken for a CV to complete a cycle was twice as long for a P2XA − vacuole compared to wild-type vacuoles. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 vacuoles. Statistical source data for Fig 2E can be found in Supplementary Table 2 .F. P2XA -cells have larger numbers of small vacuoles than wild-type cells, and very few bigger vacuoles. P2XA − cells overexpressing P2XA:GFP have more small vacuoles than wild-type cells, but the number of bigger vacuoles is similar to wild-type cells. Error bars represent s.e.m. and results are means of n=3 independent experiments, each with 50 cells. Statistical source data for Fig 2F can be found in Supplementary Table 2 .G. Zoom of fluorescence images of P2XA − cell from panel A expressing Dajumin-GFP. White arrows indicate a “budding” vacuole.

    Techniques Used: Fluorescence, Expressing

    P2XA − cells fail to undergo the ring-to-patch transition stage of the CV cycle A. Fluorescence images of a wild-type and P2XA − cells expressing Drainin-GFP. Closed white arrows indicate vacuoles at the ‘ring to patch’ transition stage. In P2XA -cells, Drainin-GFP becomes recruited to CV’s as they mature. However, Drainin-GFP localization does not change and ‘ring to patch’ transition is not observed. Open arrows indicate vacuoles that have reached the stage where ‘ring to patch’ transition should occur. B. Fluorescence images of wild-type and P2XA − cells expressing Rab8a-GFP. Closed white arrows indicate a vacuole at the ‘ring to patch’ transition stage. In P2XA − cells, Rab8a-GFP becomes recruited to mature CV’s. However, Rab8a-GFP localization does not change and ‘ring to patch’ transition is not observed. Numbers represent the time in seconds after changing the media from KK2 to water. Scale bars = 5μm.
    Figure Legend Snippet: P2XA − cells fail to undergo the ring-to-patch transition stage of the CV cycle A. Fluorescence images of a wild-type and P2XA − cells expressing Drainin-GFP. Closed white arrows indicate vacuoles at the ‘ring to patch’ transition stage. In P2XA -cells, Drainin-GFP becomes recruited to CV’s as they mature. However, Drainin-GFP localization does not change and ‘ring to patch’ transition is not observed. Open arrows indicate vacuoles that have reached the stage where ‘ring to patch’ transition should occur. B. Fluorescence images of wild-type and P2XA − cells expressing Rab8a-GFP. Closed white arrows indicate a vacuole at the ‘ring to patch’ transition stage. In P2XA − cells, Rab8a-GFP becomes recruited to mature CV’s. However, Rab8a-GFP localization does not change and ‘ring to patch’ transition is not observed. Numbers represent the time in seconds after changing the media from KK2 to water. Scale bars = 5μm.

    Techniques Used: Fluorescence, Expressing

    GCaMP2 sensor indicates that P2XA activity is increased in docked vesicles A. Currents evoked by ATP (black bar, concentrations indicated) in HEK cells expressing P2XA ( left ) or P2XA-GCaMP2 ( right ) receptors. B. Concentration-response curves for HEK cells expressing wild type (black) or (green) P2XAGCaMP2 receptors shows no significant difference in sensitivity to ATP. Error bars represent s.e.m. and results are means from n=21 wild type and n=7 P2XA-GCaMP expressing cells. C. Concentration-response curve for GCaMP2 fluorescence in HEK cells expressing P2XA-GCaMP2. Ordinate is normalized to the maximal fluorescence observed with ATP. Effective concentrations of ATP are similar to those in B (Error bars represent s.e.m. and results are means from n=7 cells). D. Line scans from HEK cells expressing P2XA-GCaMP2 at 0, 20 and 50 s after ATP application shows increased fluorescence at plasma membrane. Scale bar = 10 μm. E. ATP (1 mM) increases GCaMP2 fluorescence in cells expressing wild type P2XA receptors (filled green squares), but not in cells expressing P2XA(K67A/K289A) receptors (open green squares) or P2XA-GFP receptors (black squares). Error bars represent s.e.m. and results are means from n=6 cells). F. Dictyostelium cells co-expressing P2XA-RFP and either P2XA-GCaMP2 (filled green squares), P2XA(K67A/K289A)-GCaMP2 (open green squares), or P2XA-GFP (filled black squares) were subjected to osmotic shock. The ratio of GFP to RFP fluorescence was measured at three stages throughout the cycle (maturing, docked, and ring-to-patch) for each strain (except cells expressing P2XA(K67A/K289A)-GCaMP2 receptors were not studied at ring-to-patch stage because these cells do not undergo that stage). The GFP/RFP ratio in cells expressing P2XA-GCaMP2 increased significantly at the later stages of the CV cycle (paired t test between P2XA-GCaMP2/ P2XA-RFP and P2XA-GFP/P2XA-RFP gives p
    Figure Legend Snippet: GCaMP2 sensor indicates that P2XA activity is increased in docked vesicles A. Currents evoked by ATP (black bar, concentrations indicated) in HEK cells expressing P2XA ( left ) or P2XA-GCaMP2 ( right ) receptors. B. Concentration-response curves for HEK cells expressing wild type (black) or (green) P2XAGCaMP2 receptors shows no significant difference in sensitivity to ATP. Error bars represent s.e.m. and results are means from n=21 wild type and n=7 P2XA-GCaMP expressing cells. C. Concentration-response curve for GCaMP2 fluorescence in HEK cells expressing P2XA-GCaMP2. Ordinate is normalized to the maximal fluorescence observed with ATP. Effective concentrations of ATP are similar to those in B (Error bars represent s.e.m. and results are means from n=7 cells). D. Line scans from HEK cells expressing P2XA-GCaMP2 at 0, 20 and 50 s after ATP application shows increased fluorescence at plasma membrane. Scale bar = 10 μm. E. ATP (1 mM) increases GCaMP2 fluorescence in cells expressing wild type P2XA receptors (filled green squares), but not in cells expressing P2XA(K67A/K289A) receptors (open green squares) or P2XA-GFP receptors (black squares). Error bars represent s.e.m. and results are means from n=6 cells). F. Dictyostelium cells co-expressing P2XA-RFP and either P2XA-GCaMP2 (filled green squares), P2XA(K67A/K289A)-GCaMP2 (open green squares), or P2XA-GFP (filled black squares) were subjected to osmotic shock. The ratio of GFP to RFP fluorescence was measured at three stages throughout the cycle (maturing, docked, and ring-to-patch) for each strain (except cells expressing P2XA(K67A/K289A)-GCaMP2 receptors were not studied at ring-to-patch stage because these cells do not undergo that stage). The GFP/RFP ratio in cells expressing P2XA-GCaMP2 increased significantly at the later stages of the CV cycle (paired t test between P2XA-GCaMP2/ P2XA-RFP and P2XA-GFP/P2XA-RFP gives p

    Techniques Used: Activity Assay, Expressing, Concentration Assay, Fluorescence

    cnrF is required for normal osmoregulation and regulation of Rab11 activity A. cnrF mutant cells exhibit impaired osmoregulation. Representative bright-field images of cells in KK2, and after 10 min and 60 min after changing the media from KK2 to water to induce osmotic shock. Scale bar = 5μm. B. Time course of cell rounding and recovery. cnrF − , cnrF (R270A) and cnrF (E623Q/D659Q) mutant cells exhibit similar osmoregulation defects to P2XA − cells. Error bars represent s.e.m. from n=3 three independent experiments, each with 100 cells. Statistical source data for Fig 6B can be found in Supplementary Table 2 . C. Visualisation of CV morphology in wild-type, P2XA − cnrF − , cnrF (R270A) or cnrF (E623Q/D659Q) cells expressing Dajumin-GFP after osmotic shock .All cnrF mutants contain many irregularly sized vacuoles at the cell surface that fail to fuse. Scale bar = 5μm. D and E. Immunoprecipitation of Rab11a-GTP from wild-type, P2XA − cnrF − , cnrF (R270A), cnrF (E623Q/D659Q) and CnrF-GFP overexpressing cells expressing Rab11a-RFP (I = input, B = bound, NB = not bound). The level of Rab11a-RFP expression (input) is comparable between all strains ( > 1.3 fold difference between highest and lowest). E. Quantification of GTP and GDP bound Rab11a revealed that all mutant strains exhibit a significant (paired T test p
    Figure Legend Snippet: cnrF is required for normal osmoregulation and regulation of Rab11 activity A. cnrF mutant cells exhibit impaired osmoregulation. Representative bright-field images of cells in KK2, and after 10 min and 60 min after changing the media from KK2 to water to induce osmotic shock. Scale bar = 5μm. B. Time course of cell rounding and recovery. cnrF − , cnrF (R270A) and cnrF (E623Q/D659Q) mutant cells exhibit similar osmoregulation defects to P2XA − cells. Error bars represent s.e.m. from n=3 three independent experiments, each with 100 cells. Statistical source data for Fig 6B can be found in Supplementary Table 2 . C. Visualisation of CV morphology in wild-type, P2XA − cnrF − , cnrF (R270A) or cnrF (E623Q/D659Q) cells expressing Dajumin-GFP after osmotic shock .All cnrF mutants contain many irregularly sized vacuoles at the cell surface that fail to fuse. Scale bar = 5μm. D and E. Immunoprecipitation of Rab11a-GTP from wild-type, P2XA − cnrF − , cnrF (R270A), cnrF (E623Q/D659Q) and CnrF-GFP overexpressing cells expressing Rab11a-RFP (I = input, B = bound, NB = not bound). The level of Rab11a-RFP expression (input) is comparable between all strains ( > 1.3 fold difference between highest and lowest). E. Quantification of GTP and GDP bound Rab11a revealed that all mutant strains exhibit a significant (paired T test p

    Techniques Used: Activity Assay, Mutagenesis, Expressing, Immunoprecipitation

    26) Product Images from "Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin"

    Article Title: Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-167

    The C-terminal end of the NS1 protein in H3N2 type influenza A viruses encode for a functional NoLS. ( A ) The N- and C-terminal fragments of the NS1 genes, encoding for amino acids 1–73 and 157–237 in A/Udorn/72, amino acids 1–73 and 157–230 in A/WSN/33 and amino acids 203–230 in A/Brevig Mission/1/18, were inserted into the GFP expression vector pCMX-SAH/Y145F to express GFP-NS1 fusion proteins. Mutations in the C-terminal NLS2/NoLS of A/Udorn/72 virus NS1 protein were performed as described in Materials and Methods. Red boxes indicate the positions of N-terminal NLS1 and C-terminal NLS2/NoLS in NS1 protein. ( B ) HuH7 cells were transiently transfected with GFP, GFP-NS1 fusion and GFP-NS1 fusion mutant (K219A,R220A + R231A,R232A) A/Udorn/72 gene constructs, as indicated in the figure, for 48 h. The intensity of nucleolar localization was scored by immunofluorescence microscopy as no nucleolar staining (−) or weak (+), moderate (++) or strong (+++) nucleolar staining. Critical basic amino acids involved in nuclear/nucleolar targeting are marked in boldface and underlined. Mutated amino acids (K219A,R220A + R231A,R232A) are marked in red. ( C ) HuH7 cells were transiently transfected with GFP and the GFP-deletion gene constructs of NS1 A/WSN/33 and NS1 A/Brevig Mission/1/18 for 48 h as indicated in the figure. Critical and mutated amino acids are marked as above. Bars, 5 μm.
    Figure Legend Snippet: The C-terminal end of the NS1 protein in H3N2 type influenza A viruses encode for a functional NoLS. ( A ) The N- and C-terminal fragments of the NS1 genes, encoding for amino acids 1–73 and 157–237 in A/Udorn/72, amino acids 1–73 and 157–230 in A/WSN/33 and amino acids 203–230 in A/Brevig Mission/1/18, were inserted into the GFP expression vector pCMX-SAH/Y145F to express GFP-NS1 fusion proteins. Mutations in the C-terminal NLS2/NoLS of A/Udorn/72 virus NS1 protein were performed as described in Materials and Methods. Red boxes indicate the positions of N-terminal NLS1 and C-terminal NLS2/NoLS in NS1 protein. ( B ) HuH7 cells were transiently transfected with GFP, GFP-NS1 fusion and GFP-NS1 fusion mutant (K219A,R220A + R231A,R232A) A/Udorn/72 gene constructs, as indicated in the figure, for 48 h. The intensity of nucleolar localization was scored by immunofluorescence microscopy as no nucleolar staining (−) or weak (+), moderate (++) or strong (+++) nucleolar staining. Critical basic amino acids involved in nuclear/nucleolar targeting are marked in boldface and underlined. Mutated amino acids (K219A,R220A + R231A,R232A) are marked in red. ( C ) HuH7 cells were transiently transfected with GFP and the GFP-deletion gene constructs of NS1 A/WSN/33 and NS1 A/Brevig Mission/1/18 for 48 h as indicated in the figure. Critical and mutated amino acids are marked as above. Bars, 5 μm.

    Techniques Used: Functional Assay, Expressing, Plasmid Preparation, Transfection, Mutagenesis, Construct, Immunofluorescence, Microscopy, Staining

    Chimeric GFP-NS1 A/Udorn/72/(203–237) colocalizes with HIV-1 Rev protein in the nucleolus, and a high expression level of HIV-1 Rev protein totally displaced the GFP-NS1(203–237) fusion protein from the nucleoli to the nucleus. HuH7 cells were grown on coverslips for 24 h and then transiently transfected with HIV-1 Rev and chimeric GFP-NS1 A/Udorn/72/(203–237) gene constructs for 48 h. After fixation, the cells were stained with rabbit anti-HIV-1 Rev and Rhodamine Red-X -labeled goat anti-rabbit immunoglobulins, followed by analysis with confocal laser microscopy. A high expression level of HIV-1 Rev protein totally displaced the GFP-NS1(203–237) fusion protein from the nucleoli to the nucleus (bottom panels). Cells, expressing low, medium or high amounts of nucleolar HIV-1 Rev protein, were selected. Only the nuclei of the cells are presented. Bar, 5 μm.
    Figure Legend Snippet: Chimeric GFP-NS1 A/Udorn/72/(203–237) colocalizes with HIV-1 Rev protein in the nucleolus, and a high expression level of HIV-1 Rev protein totally displaced the GFP-NS1(203–237) fusion protein from the nucleoli to the nucleus. HuH7 cells were grown on coverslips for 24 h and then transiently transfected with HIV-1 Rev and chimeric GFP-NS1 A/Udorn/72/(203–237) gene constructs for 48 h. After fixation, the cells were stained with rabbit anti-HIV-1 Rev and Rhodamine Red-X -labeled goat anti-rabbit immunoglobulins, followed by analysis with confocal laser microscopy. A high expression level of HIV-1 Rev protein totally displaced the GFP-NS1(203–237) fusion protein from the nucleoli to the nucleus (bottom panels). Cells, expressing low, medium or high amounts of nucleolar HIV-1 Rev protein, were selected. Only the nuclei of the cells are presented. Bar, 5 μm.

    Techniques Used: Expressing, Transfection, Construct, Staining, Labeling, Microscopy

    27) Product Images from "Narrow-Leafed Lupin (Lupinus angustifolius) β1- and β6-Conglutin Proteins Exhibit Antifungal Activity, Protecting Plants against Necrotrophic Pathogen Induced Damage from Sclerotinia sclerotiorum and Phytophthora nicotianae"

    Article Title: Narrow-Leafed Lupin (Lupinus angustifolius) β1- and β6-Conglutin Proteins Exhibit Antifungal Activity, Protecting Plants against Necrotrophic Pathogen Induced Damage from Sclerotinia sclerotiorum and Phytophthora nicotianae

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.01856

    Recombinant β6-conglutin exhibits in planta anti-fungal and oomycete activity. Shown are representative images of Agrobacterium infiltrated N. benthamiana leaves expressing recombinant β6-conglutin proteins and subsequently inoculated with either S. sclerotiorum or P. nicotianae . The experiment was repeated three times with similar results. C, control agroinfiltration of the leaf area with Agrobacterium expressing GFP only; β6, agroinfiltration of the leaf area with Agrobacterium expressing GFP tagged β6-conglutin.
    Figure Legend Snippet: Recombinant β6-conglutin exhibits in planta anti-fungal and oomycete activity. Shown are representative images of Agrobacterium infiltrated N. benthamiana leaves expressing recombinant β6-conglutin proteins and subsequently inoculated with either S. sclerotiorum or P. nicotianae . The experiment was repeated three times with similar results. C, control agroinfiltration of the leaf area with Agrobacterium expressing GFP only; β6, agroinfiltration of the leaf area with Agrobacterium expressing GFP tagged β6-conglutin.

    Techniques Used: Recombinant, Activity Assay, Expressing

    β6-conglutin localizes to the plasmodesmata. Single-slice confocal images of co-expression GFP-β6 with the plasmodesmata marker PDLP1-mCherry after transient expression in N. benthamiana ; (A) PPDLP1-mCherry, (B) β6-GFP, (C) Image showing pixel pairs that have a positive PDM value equal to the value (intensity of A- mean A intensity) ∗ (intensity of B-mean B intensity) as described in Li et al. (2004) , (D) merge of (A,B) with highlighted co-localized pixels. ICQ, Intensity correlation quotient; R, Mandel’s overlap coefficient. 60× immersion objective.
    Figure Legend Snippet: β6-conglutin localizes to the plasmodesmata. Single-slice confocal images of co-expression GFP-β6 with the plasmodesmata marker PDLP1-mCherry after transient expression in N. benthamiana ; (A) PPDLP1-mCherry, (B) β6-GFP, (C) Image showing pixel pairs that have a positive PDM value equal to the value (intensity of A- mean A intensity) ∗ (intensity of B-mean B intensity) as described in Li et al. (2004) , (D) merge of (A,B) with highlighted co-localized pixels. ICQ, Intensity correlation quotient; R, Mandel’s overlap coefficient. 60× immersion objective.

    Techniques Used: Expressing, Marker

    β6-conglutin is localized to the cell surface. Confocal images of tobacco epidermis cell expressing GFP alone or β6-GFP shows GFP alone homogenously expressed throughout the cytoplasm while β6-conglutin localizes to the plasma membrane through the whole cell, with no expression in cytosol or intracellular organelles. Insert: β6-GFP shows punctate labeling at the cell surface.
    Figure Legend Snippet: β6-conglutin is localized to the cell surface. Confocal images of tobacco epidermis cell expressing GFP alone or β6-GFP shows GFP alone homogenously expressed throughout the cytoplasm while β6-conglutin localizes to the plasma membrane through the whole cell, with no expression in cytosol or intracellular organelles. Insert: β6-GFP shows punctate labeling at the cell surface.

    Techniques Used: Expressing, Labeling

    β6-conglutin oxyblots assayes. Protein carbonyl formation in tobacco leaves 48 h after agroinfiltration with indicated constructs and 24 h after inoculation with pathogens. Protein carbonyls were assessed using an OxyBlot TM kit. (A) Typical levels of pathogen growth 24 h after inoculation of leaf samples used for assay. (B) Representative blot showing basal carbonylation levels in non-transformed leaves, control leaves expressing GFP mock-inoculated and after infection with P. nicotianae , and leaves expressing β6-GFP mock-inoculated and after infection. (C) Protein carbonylation levels after infection with S. sclerotiorum , same constructs as in (B) .
    Figure Legend Snippet: β6-conglutin oxyblots assayes. Protein carbonyl formation in tobacco leaves 48 h after agroinfiltration with indicated constructs and 24 h after inoculation with pathogens. Protein carbonyls were assessed using an OxyBlot TM kit. (A) Typical levels of pathogen growth 24 h after inoculation of leaf samples used for assay. (B) Representative blot showing basal carbonylation levels in non-transformed leaves, control leaves expressing GFP mock-inoculated and after infection with P. nicotianae , and leaves expressing β6-GFP mock-inoculated and after infection. (C) Protein carbonylation levels after infection with S. sclerotiorum , same constructs as in (B) .

    Techniques Used: Construct, Transformation Assay, Expressing, Infection

    28) Product Images from "Structural basis for misregulation of kinesin KIF21A autoinhibition by CFEOM1 disease mutations"

    Article Title: Structural basis for misregulation of kinesin KIF21A autoinhibition by CFEOM1 disease mutations

    Journal: Scientific Reports

    doi: 10.1038/srep30668

    The regulatory antiparallel coiled coil binds to the KIF21A and KIF21B motor domains and is sufficient for KIF21A autoinhibition. ( a,b ) Streptavidin-based pull-down assay with lysates of HEK293T cells expressing the indicated proteins. Bio-GFP-tagged KIF21A_MD2 ( a ) and Bio-GFP-tagged KIF21B_MD1 ( b ) were used as baits. Coiled-coil polypeptide chain fragments are mCherry-tagged. ( c ) Binding affinity determined by ITC. rCC1 at a protein concentration of 900 μM was titrated into 48 μM solution of KIF21B_MD in 20 mM TrisHCl pH 7.5, 150 mM NaCl and 2 mM DTT and heat was measured at 10 °C. The ITC thermograph and fitted binding isotherm are shown in the upper and lower panel, respectively. ( d ) In vitro kinesin motility assay. Purified KIF21A MD2-GFP was added to taxol-stabilized MTs in flow chambers and analyzed for MT association in the presence of GCN4, rCC1 or rCC1-L. Both short MT binding and motile events were counted. Two independent measurements were analysed for rCC1, while 3 measurements were included for GCN4 and rCC1-L. (*P
    Figure Legend Snippet: The regulatory antiparallel coiled coil binds to the KIF21A and KIF21B motor domains and is sufficient for KIF21A autoinhibition. ( a,b ) Streptavidin-based pull-down assay with lysates of HEK293T cells expressing the indicated proteins. Bio-GFP-tagged KIF21A_MD2 ( a ) and Bio-GFP-tagged KIF21B_MD1 ( b ) were used as baits. Coiled-coil polypeptide chain fragments are mCherry-tagged. ( c ) Binding affinity determined by ITC. rCC1 at a protein concentration of 900 μM was titrated into 48 μM solution of KIF21B_MD in 20 mM TrisHCl pH 7.5, 150 mM NaCl and 2 mM DTT and heat was measured at 10 °C. The ITC thermograph and fitted binding isotherm are shown in the upper and lower panel, respectively. ( d ) In vitro kinesin motility assay. Purified KIF21A MD2-GFP was added to taxol-stabilized MTs in flow chambers and analyzed for MT association in the presence of GCN4, rCC1 or rCC1-L. Both short MT binding and motile events were counted. Two independent measurements were analysed for rCC1, while 3 measurements were included for GCN4 and rCC1-L. (*P

    Techniques Used: Pull Down Assay, Expressing, Binding Assay, Protein Concentration, In Vitro, Motility Assay, Purification, Flow Cytometry

    The intramolecular antiparallel coiled coil is an autonomous folding unit within the dimeric KIF21A stalk. ( a ) Distribution of initial (non-bleached) fluorescent intensities of single molecules. TIRF single molecule imaging was performed using HEK293T-cell extracts expressing indicated N-terminally biotinylated (Bio) GFP-tagged constructs. GFP and EB1-GFP serve as monomeric and dimeric controls, respectively 20 . ( b ) Schematic representation of two possible coiled-coil conformations of the KIF21A regulatory domain (dark grey) in the context of the KIF21A STALK (light grey). In the parallel conformation, Cys1006 (red) would be crosslinked to Cys1006′ of the opposite helix. In an intramolecular antiparallel conformation, Cys1006 would not be crosslinked. Amino (N) and carboxy (C) termini are indicated. ( c ) Cysteine crosslinking of GFP-labelled CLIP-115 and KIF21A stalk-domain fragments analyzed under reducing and non-reducing conditions by SDS-PAGE.
    Figure Legend Snippet: The intramolecular antiparallel coiled coil is an autonomous folding unit within the dimeric KIF21A stalk. ( a ) Distribution of initial (non-bleached) fluorescent intensities of single molecules. TIRF single molecule imaging was performed using HEK293T-cell extracts expressing indicated N-terminally biotinylated (Bio) GFP-tagged constructs. GFP and EB1-GFP serve as monomeric and dimeric controls, respectively 20 . ( b ) Schematic representation of two possible coiled-coil conformations of the KIF21A regulatory domain (dark grey) in the context of the KIF21A STALK (light grey). In the parallel conformation, Cys1006 (red) would be crosslinked to Cys1006′ of the opposite helix. In an intramolecular antiparallel conformation, Cys1006 would not be crosslinked. Amino (N) and carboxy (C) termini are indicated. ( c ) Cysteine crosslinking of GFP-labelled CLIP-115 and KIF21A stalk-domain fragments analyzed under reducing and non-reducing conditions by SDS-PAGE.

    Techniques Used: Imaging, Expressing, Construct, Cross-linking Immunoprecipitation, SDS Page

    CFEOM1 mutations release KIF21A autoinhibition by destabilizing the regulatory antiparallel coiled coil or by affecting residues of the binding interface. ( a ) Positions of the CFEOM1-associated amino-acid residues shown in one half of the KIF21A rCC1 crystal structure. Amino (N) and carboxy (C) termini are indicated. ( b ) Summary of the biophysical characterization of wild-type rCC1-L and its CFEOM1 mutants by thermal unfolding monitored by CD at 222 nm using a protein concentration of 0.125 mg/ml and SEC-MALS performed at a protein concentration of 2 mg/ml. ( c ) Normalized thermal unfolding profiles of wild-type rCC1-L and its most destabilizing (M947T) and most stabilizing mutants (R954L). ( d ) Streptavidin-based pull-down assay with lysates of HEK293T cells expressing Bio-GFP-tagged KIF21A_MD2 used as bait for the pulldown of mCherry-labelled STALK1_WT and mutant polypeptide chain fragments. ( e ) In vitro kinesin motility assay. The assay was performed as described in Fig. 5d , but in the presence of wild-type or mutant rCC1-L domains. Values significantly different from the wild-type control are indicated by an asterisk (P
    Figure Legend Snippet: CFEOM1 mutations release KIF21A autoinhibition by destabilizing the regulatory antiparallel coiled coil or by affecting residues of the binding interface. ( a ) Positions of the CFEOM1-associated amino-acid residues shown in one half of the KIF21A rCC1 crystal structure. Amino (N) and carboxy (C) termini are indicated. ( b ) Summary of the biophysical characterization of wild-type rCC1-L and its CFEOM1 mutants by thermal unfolding monitored by CD at 222 nm using a protein concentration of 0.125 mg/ml and SEC-MALS performed at a protein concentration of 2 mg/ml. ( c ) Normalized thermal unfolding profiles of wild-type rCC1-L and its most destabilizing (M947T) and most stabilizing mutants (R954L). ( d ) Streptavidin-based pull-down assay with lysates of HEK293T cells expressing Bio-GFP-tagged KIF21A_MD2 used as bait for the pulldown of mCherry-labelled STALK1_WT and mutant polypeptide chain fragments. ( e ) In vitro kinesin motility assay. The assay was performed as described in Fig. 5d , but in the presence of wild-type or mutant rCC1-L domains. Values significantly different from the wild-type control are indicated by an asterisk (P

    Techniques Used: Binding Assay, Protein Concentration, Size-exclusion Chromatography, Pull Down Assay, Expressing, Mutagenesis, In Vitro, Motility Assay

    Molecular docking of the KIF21A motor domain and the regulatory antiparallel coiled coil. ( a ) KIF21A homology model based on the KIF4 motor-domain structure (PDB code 3ZFD). The positions of CFEOM1 mutations in the motor domain are indicated. The α-tubulin subunit interacting elements are labeled in blue on the basis of the KIF5-tubulin complex crystal structure (PDB code 4HNA 30 ). ( b ) Docking model (light grey) with residues targeted for mutagenesis highlighted. The NKQ residues involved in the interface and the DYD residues pointing away from the interface (negative control) are shown in orange and green, respectively. ( c–e ) TIRFM-based live cell imaging of COS-7 cells transiently transfected with KIF21A_MD3 or the two mutant versions of this construct, NKQ or DYD, fused to GFP. Panels from left to right: a single frame of the GFP channel; maximum intensity projection of the GFP channel over 500 frames (50 s), and a kymograph along a single MT, illustrating the motile behavior of KIF21A_MD3-NKQ and very limited motility of the wild-type KIF21A_MD3 and KIF21A_MD3-DYD. ( f ) Superposition of the KIF5-tubulin complex (dark green α-tubulin, light green β-tubulin) crystal structure (PDB code 4HNA 30 ) and the KIF21A docking model (grey) indicates the mechanistic details of KIF21A autoinhibition. ( g ) Sequence alignment of the regulatory antiparallel coiled coil in hsKIF21A with the corresponding sequences in hsKIF21B, hsKIF7 and hsKIF27. Conserved amino-acid residues are marked by an asterisk. Similar hydrophobic (green), polar (orange), positively charged (blue) and negatively charged (red) amino acids are depicted. Amino acids known to be mutated in patients with Bardet-Biedl syndrome are indicated by arrows. CFEOM1-associated amino acids are highlighted (grey). Heptad repeats are shown as blocks of seven residues denoted a to g .
    Figure Legend Snippet: Molecular docking of the KIF21A motor domain and the regulatory antiparallel coiled coil. ( a ) KIF21A homology model based on the KIF4 motor-domain structure (PDB code 3ZFD). The positions of CFEOM1 mutations in the motor domain are indicated. The α-tubulin subunit interacting elements are labeled in blue on the basis of the KIF5-tubulin complex crystal structure (PDB code 4HNA 30 ). ( b ) Docking model (light grey) with residues targeted for mutagenesis highlighted. The NKQ residues involved in the interface and the DYD residues pointing away from the interface (negative control) are shown in orange and green, respectively. ( c–e ) TIRFM-based live cell imaging of COS-7 cells transiently transfected with KIF21A_MD3 or the two mutant versions of this construct, NKQ or DYD, fused to GFP. Panels from left to right: a single frame of the GFP channel; maximum intensity projection of the GFP channel over 500 frames (50 s), and a kymograph along a single MT, illustrating the motile behavior of KIF21A_MD3-NKQ and very limited motility of the wild-type KIF21A_MD3 and KIF21A_MD3-DYD. ( f ) Superposition of the KIF5-tubulin complex (dark green α-tubulin, light green β-tubulin) crystal structure (PDB code 4HNA 30 ) and the KIF21A docking model (grey) indicates the mechanistic details of KIF21A autoinhibition. ( g ) Sequence alignment of the regulatory antiparallel coiled coil in hsKIF21A with the corresponding sequences in hsKIF21B, hsKIF7 and hsKIF27. Conserved amino-acid residues are marked by an asterisk. Similar hydrophobic (green), polar (orange), positively charged (blue) and negatively charged (red) amino acids are depicted. Amino acids known to be mutated in patients with Bardet-Biedl syndrome are indicated by arrows. CFEOM1-associated amino acids are highlighted (grey). Heptad repeats are shown as blocks of seven residues denoted a to g .

    Techniques Used: Labeling, Mutagenesis, Negative Control, Live Cell Imaging, Transfection, Construct, Sequencing

    29) Product Images from "Small Heat Shock Protein Hsp17.8 Functions as an AKR2A Cofactor in the Targeting of Chloroplast Outer Membrane Proteins in Arabidopsis 1Small Heat Shock Protein Hsp17.8 Functions as an AKR2A Cofactor in the Targeting of Chloroplast Outer Membrane Proteins in Arabidopsis 1 [W]Small Heat Shock Protein Hsp17.8 Functions as an AKR2A Cofactor in the Targeting of Chloroplast Outer Membrane Proteins in Arabidopsis 1 [W] [OA]"

    Article Title: Small Heat Shock Protein Hsp17.8 Functions as an AKR2A Cofactor in the Targeting of Chloroplast Outer Membrane Proteins in Arabidopsis 1Small Heat Shock Protein Hsp17.8 Functions as an AKR2A Cofactor in the Targeting of Chloroplast Outer Membrane Proteins in Arabidopsis 1 [W]Small Heat Shock Protein Hsp17.8 Functions as an AKR2A Cofactor in the Targeting of Chloroplast Outer Membrane Proteins in Arabidopsis 1 [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.111.178681

    Hsp17.8 increases the targeting efficiency of OEP7:GFP to chloroplasts. A and B, The effect of Hsp17.8 on OEP7:GFP targeting to chloroplasts. A, Protoplasts were cotransformed with the indicated constructs, and proteins from the transformed protoplasts
    Figure Legend Snippet: Hsp17.8 increases the targeting efficiency of OEP7:GFP to chloroplasts. A and B, The effect of Hsp17.8 on OEP7:GFP targeting to chloroplasts. A, Protoplasts were cotransformed with the indicated constructs, and proteins from the transformed protoplasts

    Techniques Used: Construct, Transformation Assay

    Hsp17.8 does not bind directly to OEP7 in the cytoplasm. GFP : OEP7 was introduced into protoplasts together with HA : AKR2A , HSP17.8 : HA , or R6 , and the GFP pattern of GFP:OEP7 was examined. Bottom panels show bright-field images. CH, Chloroplasts. Bars =
    Figure Legend Snippet: Hsp17.8 does not bind directly to OEP7 in the cytoplasm. GFP : OEP7 was introduced into protoplasts together with HA : AKR2A , HSP17.8 : HA , or R6 , and the GFP pattern of GFP:OEP7 was examined. Bottom panels show bright-field images. CH, Chloroplasts. Bars =

    Techniques Used:

    30) Product Images from "Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to DiminishS-Adenosylmethionine Synthesis *-Adenosylmethionine Synthesis * S⃞"

    Article Title: Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to DiminishS-Adenosylmethionine Synthesis *-Adenosylmethionine Synthesis * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804159200

    MAT-IIβ ablation diminishes leukemic cell growth in vivo . Hyperimmune-deficient NOD/Scid IL-2Rγ null mice were irradiated with 3.5GY 24 h prior to intraperitoneal transplant with 15 × 10 6 of the indicated cells. Controls included irradiated but not injected mice and V1302 transplanted mice. Tumor engraftment and growth were monitored by measuring % human CD3 + /GFP + cells in mice spleens and bone marrow at 4 and 5 weeks post-transplant with either control V1302 or V1110 cells. Flow cytometry histograms of CD3 + /GFP + in spleen ( A ) or bone marrow ( B ) 5 weeks post-transplant. C , GFP expression in whole spleens of same mice prior to processing their splenocytes. D , number of mice engrafted with V1302 or V1110 leukemic cells at 4 and 5 weeks post-transplant. E and F show % CD3 + /GFP + cells in spleens ( E ) or bone marrow ( F ) of mice transplanted with either V1302 or V1110 leukemic cells, 5 weeks post-transplant. The statistical differences were calculated using a Mann-Whitney test. * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001.
    Figure Legend Snippet: MAT-IIβ ablation diminishes leukemic cell growth in vivo . Hyperimmune-deficient NOD/Scid IL-2Rγ null mice were irradiated with 3.5GY 24 h prior to intraperitoneal transplant with 15 × 10 6 of the indicated cells. Controls included irradiated but not injected mice and V1302 transplanted mice. Tumor engraftment and growth were monitored by measuring % human CD3 + /GFP + cells in mice spleens and bone marrow at 4 and 5 weeks post-transplant with either control V1302 or V1110 cells. Flow cytometry histograms of CD3 + /GFP + in spleen ( A ) or bone marrow ( B ) 5 weeks post-transplant. C , GFP expression in whole spleens of same mice prior to processing their splenocytes. D , number of mice engrafted with V1302 or V1110 leukemic cells at 4 and 5 weeks post-transplant. E and F show % CD3 + /GFP + cells in spleens ( E ) or bone marrow ( F ) of mice transplanted with either V1302 or V1110 leukemic cells, 5 weeks post-transplant. The statistical differences were calculated using a Mann-Whitney test. * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001.

    Techniques Used: In Vivo, Mouse Assay, Irradiation, Injection, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

    31) Product Images from "ETO/MTG8 Is an Inhibitor of C/EBP? Activity and a Regulator of Early Adipogenesis"

    Article Title: ETO/MTG8 Is an Inhibitor of C/EBP? Activity and a Regulator of Early Adipogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9863-9872.2004

    ETO impairs the induction of C/EBPα but not C/EBPβ or C/EBPδ during adipogenesis. 3T3-L1 cells expressing GFP or GFP-ETO were differentiated for the times shown. Cell lysates were prepared and analyzed by Western blotting to determine the expression of C/EBPβ, C/EBPδ or C/EBPα as indicated.
    Figure Legend Snippet: ETO impairs the induction of C/EBPα but not C/EBPβ or C/EBPδ during adipogenesis. 3T3-L1 cells expressing GFP or GFP-ETO were differentiated for the times shown. Cell lysates were prepared and analyzed by Western blotting to determine the expression of C/EBPβ, C/EBPδ or C/EBPα as indicated.

    Techniques Used: Expressing, Western Blot

    ETO localizes to the nucleus and inhibits adipogenesis, whereas the mutant ETO-AA, in which the nuclear localization signal is disrupted, is targeted to the cytosol and accelerates preadipocyte differentiation. (A) The construction of N terminally GFP-tagged forms of ETO is shown. The nuclear localization sequence (NLS) was disrupted by the introduction of mutations at codons 238 and 239 to generate GFP-tagged ETO-AA. The positions of the NHR domains, involved in protein-protein interactions, are indicated. (B) GFP-ETO (G-ETO-WT) and GFP-ETO-AA were expressed in HepG2 cells in the presence or absence of untagged wild-type ETO (ETO) as indicated. ETO proteins were analyzed by Western blotting in whole-cell lysates (upper panel) or anti-GFP immunoprecipitates obtained by using agarose conjugated anti-GFP polyclonal antibody (lower panel). (C) Subconfluent cultures of 3T3-L1 preadipocytes infected with retroviruses encoding GFP alone (mock) or GFP-tagged ETO (ETO-WT) or ETO-AA were fixed in 4% paraformaldehyde and fluorescent images captured by laser-scanning confocal microscopy. (D) Two-day postconfluent 3T3-L1 preadipocytes retrovirally transfected with GFP (m), GFP-ETO (E), or GFP-ETO-AA (AA) were treated for the times indicated in the absence or presence of differentiation mixture as indicated. RNA was extracted and ETO mRNA expression determined by Northern blotting. E, 3T3-L1 cells expressing GFP, GFP-ETO or GFP-ETO-AA were differentiated for 8 days and lipid accumulation assessed by oil-red O staining (upper panels) or light microscopy (lower panels).
    Figure Legend Snippet: ETO localizes to the nucleus and inhibits adipogenesis, whereas the mutant ETO-AA, in which the nuclear localization signal is disrupted, is targeted to the cytosol and accelerates preadipocyte differentiation. (A) The construction of N terminally GFP-tagged forms of ETO is shown. The nuclear localization sequence (NLS) was disrupted by the introduction of mutations at codons 238 and 239 to generate GFP-tagged ETO-AA. The positions of the NHR domains, involved in protein-protein interactions, are indicated. (B) GFP-ETO (G-ETO-WT) and GFP-ETO-AA were expressed in HepG2 cells in the presence or absence of untagged wild-type ETO (ETO) as indicated. ETO proteins were analyzed by Western blotting in whole-cell lysates (upper panel) or anti-GFP immunoprecipitates obtained by using agarose conjugated anti-GFP polyclonal antibody (lower panel). (C) Subconfluent cultures of 3T3-L1 preadipocytes infected with retroviruses encoding GFP alone (mock) or GFP-tagged ETO (ETO-WT) or ETO-AA were fixed in 4% paraformaldehyde and fluorescent images captured by laser-scanning confocal microscopy. (D) Two-day postconfluent 3T3-L1 preadipocytes retrovirally transfected with GFP (m), GFP-ETO (E), or GFP-ETO-AA (AA) were treated for the times indicated in the absence or presence of differentiation mixture as indicated. RNA was extracted and ETO mRNA expression determined by Northern blotting. E, 3T3-L1 cells expressing GFP, GFP-ETO or GFP-ETO-AA were differentiated for 8 days and lipid accumulation assessed by oil-red O staining (upper panels) or light microscopy (lower panels).

    Techniques Used: Mutagenesis, Sequencing, Western Blot, Infection, Confocal Microscopy, Transfection, Expressing, Northern Blot, Staining, Light Microscopy

    ETO inhibits the expression of adipogenic proteins. Cells retrovirally transfected with GFP (▪), GFP-ETO (□) or GFP-ETO-AA ( ) were induced to differentiate for 0 to 12 days (D0 to D12) as indicated. Total cell lysates were prepared and proteins analyzed by SDS-PAGE and Western blotting with antibodies to C/EBPα (A), PPARγ (B), or aP2 (C) as appropriate. In each case band intensities were quantified from four independent experiments and the mean data ± the SEM is presented below a representative blot. The data were calculated as the percentage of expression at D12 in the mock transfected cells. Asterisks indicate a statistically significant difference from the expression in mock-transfected cells at the same time point ( P
    Figure Legend Snippet: ETO inhibits the expression of adipogenic proteins. Cells retrovirally transfected with GFP (▪), GFP-ETO (□) or GFP-ETO-AA ( ) were induced to differentiate for 0 to 12 days (D0 to D12) as indicated. Total cell lysates were prepared and proteins analyzed by SDS-PAGE and Western blotting with antibodies to C/EBPα (A), PPARγ (B), or aP2 (C) as appropriate. In each case band intensities were quantified from four independent experiments and the mean data ± the SEM is presented below a representative blot. The data were calculated as the percentage of expression at D12 in the mock transfected cells. Asterisks indicate a statistically significant difference from the expression in mock-transfected cells at the same time point ( P

    Techniques Used: Expressing, Transfection, SDS Page, Western Blot

    ETO inhibits the induction of adipogenic gene expression. RNA was isolated from cells retrovirally transfected with GFP (▪), GFP-ETO (□) or GFP-ETO-AA ( ) that had been induced to differentiate for 0 to 12 days (D0 to D8) as indicated. (A) Expression of mRNA encoding C/EBPα was determined by Northern blotting. A representative blot is shown along with 18S rRNA, which was used as a loading control and all values were adjusted accordingly. C/EBPα mRNA expression was quantified in four independent experiments, and the mean ± the SEM is shown in the lower panel. Values were expressed as percentage of those in mock-transfected cells at day 12. Asterisks indicate a statistically significant difference from mock transfected cells at the same time point ( P
    Figure Legend Snippet: ETO inhibits the induction of adipogenic gene expression. RNA was isolated from cells retrovirally transfected with GFP (▪), GFP-ETO (□) or GFP-ETO-AA ( ) that had been induced to differentiate for 0 to 12 days (D0 to D8) as indicated. (A) Expression of mRNA encoding C/EBPα was determined by Northern blotting. A representative blot is shown along with 18S rRNA, which was used as a loading control and all values were adjusted accordingly. C/EBPα mRNA expression was quantified in four independent experiments, and the mean ± the SEM is shown in the lower panel. Values were expressed as percentage of those in mock-transfected cells at day 12. Asterisks indicate a statistically significant difference from mock transfected cells at the same time point ( P

    Techniques Used: Expressing, Isolation, Transfection, Northern Blot

    ETO interacts directly with C/EBPβ inhibiting its DNA-binding activity toward the C/EBPα promoter and preventing centromeric localization during adipogenesis. (A) HEK293 cells were transfected with control vector, GFP-ETO (G-ETO-WT), or GFP-ETO-AA in the absence or presence of C/EBPβ as indicated. Anti-C/EBPβ immunoprecipitates were analyzed for associated ETO protein (upper panel), whereas corresponding cell lysates were probed for ETO (middle panel) or C/EBPβ (lower panel) by Western blotting. (B) 3T3-L1 preadipocytes expressing GFP-ETO were treated for 4 h in the absence or presence of differentiation cocktail as indicated prior to lysis. Cell lysates (left panels) or immunoprecipitates prepared by using an anti-ETO antibody (Santa Cruz Biotechnology) (right panels) were analyzed by Western blotting to detect ETO (upper panels) or C/EBPβ isoforms (lower panels). (C) In vitro transcribed/translated C/EBPβ (Cβ) and/or ETO (E) were incubated with radiolabeled DNA probe corresponding to the proximal C/EBPα binding site of the C/EBPα promoter in a gel shift assay. Various ratios of C/EBPβ to ETO were achieved by adjusting the quantity of ETO. In all lanes total protein input was kept constant by appropriate addition of rabbit reticulocyte lysate, except in lane 1, where free labeled probe was run alone. (D) 3T3-L1 preadipocytes were induced to differentiate for the times indicated and ChIP assays performed by using an anti-C/EBPβ antibody to isolate C/EBPβ-associated DNA. DNA from these immunoprecipitates corresponding to the C/EBPβ binding site in the C/EBPα promoter was quantified by using real-time PCR and normalized to DNA from a 10% sample of corresponding input lysate. The data are means ± the SEM from three independent experiments. Asterisks indicate a statistically significant difference ( P
    Figure Legend Snippet: ETO interacts directly with C/EBPβ inhibiting its DNA-binding activity toward the C/EBPα promoter and preventing centromeric localization during adipogenesis. (A) HEK293 cells were transfected with control vector, GFP-ETO (G-ETO-WT), or GFP-ETO-AA in the absence or presence of C/EBPβ as indicated. Anti-C/EBPβ immunoprecipitates were analyzed for associated ETO protein (upper panel), whereas corresponding cell lysates were probed for ETO (middle panel) or C/EBPβ (lower panel) by Western blotting. (B) 3T3-L1 preadipocytes expressing GFP-ETO were treated for 4 h in the absence or presence of differentiation cocktail as indicated prior to lysis. Cell lysates (left panels) or immunoprecipitates prepared by using an anti-ETO antibody (Santa Cruz Biotechnology) (right panels) were analyzed by Western blotting to detect ETO (upper panels) or C/EBPβ isoforms (lower panels). (C) In vitro transcribed/translated C/EBPβ (Cβ) and/or ETO (E) were incubated with radiolabeled DNA probe corresponding to the proximal C/EBPα binding site of the C/EBPα promoter in a gel shift assay. Various ratios of C/EBPβ to ETO were achieved by adjusting the quantity of ETO. In all lanes total protein input was kept constant by appropriate addition of rabbit reticulocyte lysate, except in lane 1, where free labeled probe was run alone. (D) 3T3-L1 preadipocytes were induced to differentiate for the times indicated and ChIP assays performed by using an anti-C/EBPβ antibody to isolate C/EBPβ-associated DNA. DNA from these immunoprecipitates corresponding to the C/EBPβ binding site in the C/EBPα promoter was quantified by using real-time PCR and normalized to DNA from a 10% sample of corresponding input lysate. The data are means ± the SEM from three independent experiments. Asterisks indicate a statistically significant difference ( P

    Techniques Used: Binding Assay, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Lysis, In Vitro, Incubation, Electrophoretic Mobility Shift Assay, Labeling, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    32) Product Images from "A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B"

    Article Title: A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201210033

    The microtubule-binding domain of HEC1 directs MPS1 localization and function. (A) Time-lapse analysis of duration of mitotic arrest in nocodazole- and ZM447439 (ZM)-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA and expressing the indicated GFP-HEC1 proteins. Data indicate cumulative percentages of cells (from a total of ≥125 cells per treatment) that exit mitosis (scored as cell flattening) at the indicated times after NEB and are representative of three independent experiments. (b–d) Representative images (b) and quantification (c and d) of immunolocalization of MPS1, the indicated GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in c displays total kinetochore intensities (±SEM) of the indicated proteins relative to centromeres (CREST). Data are from a total of ≥103 cells per treatment from two experiments. Ratios are set to 1 for mock RNAi–treated cells (MPS1) and for GFP-HEC1 WT –expressing cells (GFP-HEC1). Graph in d displays total kinetochore intensities of the indicated proteins relative to centromeres (CREST) for all cells of a single experiment. (e and f) Representative images (e) and quantification (f) of immunolocalization of MPS1, the indicated LacI-GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated U2OS-LacO cells. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in f displays total intensities (±SEM) of MPS1 at LacO arrays relative to LacI-GFP-HEC1 (GFP) and total intensities of LacI-GFP-HEC1. Data are from a total of ≥17 cells from two experiments. Ratios for LacI-GFP-HEC1 WT –expressing cells are set to 1. Bars, 5 µm. WT, wild type; a.u., arbitrary unit.
    Figure Legend Snippet: The microtubule-binding domain of HEC1 directs MPS1 localization and function. (A) Time-lapse analysis of duration of mitotic arrest in nocodazole- and ZM447439 (ZM)-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA and expressing the indicated GFP-HEC1 proteins. Data indicate cumulative percentages of cells (from a total of ≥125 cells per treatment) that exit mitosis (scored as cell flattening) at the indicated times after NEB and are representative of three independent experiments. (b–d) Representative images (b) and quantification (c and d) of immunolocalization of MPS1, the indicated GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in c displays total kinetochore intensities (±SEM) of the indicated proteins relative to centromeres (CREST). Data are from a total of ≥103 cells per treatment from two experiments. Ratios are set to 1 for mock RNAi–treated cells (MPS1) and for GFP-HEC1 WT –expressing cells (GFP-HEC1). Graph in d displays total kinetochore intensities of the indicated proteins relative to centromeres (CREST) for all cells of a single experiment. (e and f) Representative images (e) and quantification (f) of immunolocalization of MPS1, the indicated LacI-GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated U2OS-LacO cells. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in f displays total intensities (±SEM) of MPS1 at LacO arrays relative to LacI-GFP-HEC1 (GFP) and total intensities of LacI-GFP-HEC1. Data are from a total of ≥17 cells from two experiments. Ratios for LacI-GFP-HEC1 WT –expressing cells are set to 1. Bars, 5 µm. WT, wild type; a.u., arbitrary unit.

    Techniques Used: Binding Assay, Transfection, Expressing

    33) Product Images from "Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW"

    Article Title: Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW

    Journal: BMC Systems Biology

    doi: 10.1186/1752-0509-8-2

    Evaluation of the lower limit of Ade2 using TIPI-gTOW. (A) Growth of the cells harboring GFP- TDegF ADE2 (YSM001) with the vector and the TEV plasmid on SC plates without the indicated amino acids. Six independent colonies of each strain were tested. (B) Copy numbers of the plasmids of GFP- TDegF ADE2 (YSM001) in SC medium without the indicated amino acids. Four independent measurements were performed, and the average is shown. The error bar indicates the standard deviation. *: p
    Figure Legend Snippet: Evaluation of the lower limit of Ade2 using TIPI-gTOW. (A) Growth of the cells harboring GFP- TDegF ADE2 (YSM001) with the vector and the TEV plasmid on SC plates without the indicated amino acids. Six independent colonies of each strain were tested. (B) Copy numbers of the plasmids of GFP- TDegF ADE2 (YSM001) in SC medium without the indicated amino acids. Four independent measurements were performed, and the average is shown. The error bar indicates the standard deviation. *: p

    Techniques Used: Plasmid Preparation, Standard Deviation

    Scheme of TIPI-gTOW. We first constructed a strain in which the chromosomal target gene was replaced by a GFP- TDegF target construct. We next introduced the TEV plasmid, a plasmid for gTOW that encodes pTEV + expressed from the CUP1 promoter. According to the TIPI procedure, cleavage and rapid degradation of the GFP- TDegF target is induced by pTEV + . Using the gTOW procedure, in which the copy number of the TEV plasmid exceeds 100 under the -Leu condition, we can increase the expression of pTEV + , which accelerates the degradation of the GFP- TDegF target, reducing the level of the GFP- TDegF target. It is thus expected that the upper limit copy number of the TEV plasmid would inversely correlate with the lower limit of the GFP- TDegF target. The tug-of-war between the bias to increase the copy number of leu2d and the bias to decrease the copy number of pTEV + gene determines the plasmid copy number in the cell under the -Leu condition. It is thus possible to indirectly estimate the lower limit of the GFP- TDegF target by measuring the copy number of the TEV plasmid.
    Figure Legend Snippet: Scheme of TIPI-gTOW. We first constructed a strain in which the chromosomal target gene was replaced by a GFP- TDegF target construct. We next introduced the TEV plasmid, a plasmid for gTOW that encodes pTEV + expressed from the CUP1 promoter. According to the TIPI procedure, cleavage and rapid degradation of the GFP- TDegF target is induced by pTEV + . Using the gTOW procedure, in which the copy number of the TEV plasmid exceeds 100 under the -Leu condition, we can increase the expression of pTEV + , which accelerates the degradation of the GFP- TDegF target, reducing the level of the GFP- TDegF target. It is thus expected that the upper limit copy number of the TEV plasmid would inversely correlate with the lower limit of the GFP- TDegF target. The tug-of-war between the bias to increase the copy number of leu2d and the bias to decrease the copy number of pTEV + gene determines the plasmid copy number in the cell under the -Leu condition. It is thus possible to indirectly estimate the lower limit of the GFP- TDegF target by measuring the copy number of the TEV plasmid.

    Techniques Used: Construct, Plasmid Preparation, Expressing

    TIPI-gTOW experiments of the cell cycle regulators. Copy numbers of the plasmids in the strain; (A) P_CDC19 -500 -GFP- TdegF CDC15 (YSM002), (B) P _ CDC19 -500 - GFP- TdegF CDC20 (YSM003), and (C) P_CDC19 -600 -GFP- TdegF CDC28 (YSM005) in SC medium without the indicated amino acids. Four independent measurements were performed, and the average is shown. The error bar indicates the standard deviation. *: p
    Figure Legend Snippet: TIPI-gTOW experiments of the cell cycle regulators. Copy numbers of the plasmids in the strain; (A) P_CDC19 -500 -GFP- TdegF CDC15 (YSM002), (B) P _ CDC19 -500 - GFP- TdegF CDC20 (YSM003), and (C) P_CDC19 -600 -GFP- TdegF CDC28 (YSM005) in SC medium without the indicated amino acids. Four independent measurements were performed, and the average is shown. The error bar indicates the standard deviation. *: p

    Techniques Used: Standard Deviation

    Evaluation of the lower limits of Cdc20 protein on deletion of cell cycle regulators. (A) Relative copy number changes in the TEV plasmids in gene-deletion strains with GFP- TdegF CDC20 . Genes with previously identified genetic interactions with cdc20 mutation were boxed (summarized in Table 2 ). The original data used for the graph is shown in Table 1 . (B) Relative copy number changes in TEV plasmids in gene-deletion strains with GFP- TdegF CDC20 selected from A and simulation results. The degradation rate of kd20 is gradually increased until the cell cycle simulation indicates “dead.” In the simulation, 1: Clb1 and Clb2, 2: Clb5 and Clb6, 3: Cln1 and Cln2, and 4: Cln3 and Bck2 are implemented as single genes. 5: Swi6 is implemented into the component of SBF (together with Swi4) and MBF (together with Mbp1). The original data used for the graph is shown in Table 1 and Additional file 1 : Table S2.
    Figure Legend Snippet: Evaluation of the lower limits of Cdc20 protein on deletion of cell cycle regulators. (A) Relative copy number changes in the TEV plasmids in gene-deletion strains with GFP- TdegF CDC20 . Genes with previously identified genetic interactions with cdc20 mutation were boxed (summarized in Table 2 ). The original data used for the graph is shown in Table 1 . (B) Relative copy number changes in TEV plasmids in gene-deletion strains with GFP- TdegF CDC20 selected from A and simulation results. The degradation rate of kd20 is gradually increased until the cell cycle simulation indicates “dead.” In the simulation, 1: Clb1 and Clb2, 2: Clb5 and Clb6, 3: Cln1 and Cln2, and 4: Cln3 and Bck2 are implemented as single genes. 5: Swi6 is implemented into the component of SBF (together with Swi4) and MBF (together with Mbp1). The original data used for the graph is shown in Table 1 and Additional file 1 : Table S2.

    Techniques Used: Mutagenesis

    34) Product Images from "Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding ▿"

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding ▿

    Journal:

    doi: 10.1128/JVI.01393-07

    Validation of the cell-cell binding assay. Shown are representative flow cytometry analysis data that summarize the specificity of the binding of target Cf2Th/ELR1 to the ligand CHO cells expressing gp90-GFP under standard assay conditions. (A) Cf2Th/ELR1
    Figure Legend Snippet: Validation of the cell-cell binding assay. Shown are representative flow cytometry analysis data that summarize the specificity of the binding of target Cf2Th/ELR1 to the ligand CHO cells expressing gp90-GFP under standard assay conditions. (A) Cf2Th/ELR1

    Techniques Used: Cell Binding Assay, Flow Cytometry, Cytometry, Binding Assay, Expressing

    35) Product Images from "Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways"

    Article Title: Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways

    Journal: The EMBO Journal

    doi: 10.15252/embj.2018100276

    Hel2‐mediated K63‐linked polyubiquitination is crucial for NGD and RQC Schematic drawing of the R(CGN) 12 reporter mRNA including the two quality control pathways induced by the R(CGN) 12 translation arrest sequence. Ribosome stalling occurs during translation of the R(CGN) 12 arrest sequence (shown in orange) and induces RQC and NGD. In the RQC pathway, the stalled ribosome is dissociated into subunits, and peptidyl‐tRNA remaining on the 60S subunit is ubiquitinated by Ltn1 (shown in pink) and degraded by the proteasome. In the NGD pathway, an endonucleolytic cleavage produces two fragments, the 5′ NGD intermediate (5′ NGD‐IM) and 3′ NGD intermediate (3′ NGD‐IM). The green and thin grey lines indicate GFP and HIS3 open reading frames (ORFs), and the black line indicates an untranslated region (UTR). Schematic drawing of the truncated Hel2 mutant proteins. Activities in RQC or NGD induced by the R(CGN) 12 sequence are indicated. Western blot showing that Hel2(1–315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN) 12 reporter in ltn1 Δ cells expressing truncated Hel2 mutant protein were detected with an anti‐GFP antibody. Northern blot showing the 5′ NGD‐IM derived from the R(CGN) 12 reporter in ski2 Δ cells expressing the indicated Hel2 mutant proteins. 5′ NGD‐IMs were detected with a DIG‐labelled GFP probe. Primer extension mapping of 5′ ends of 3′ NGD intermediates in Hel2‐WT or Hel2(1–315) mutant cells at nucleotide resolution. The primer extension samples were analysed using 5% TBE‐Urea‐PAGE and detected by fluorescence. Non‐specific reverse transcription (ReTr) products are indicated by asterisks. Dissection of NGD RQC+ and NGD RQC− : the carboxyl‐terminal region of Hel2 is required for both NGD and RQC which is likely triggered on a disome unit (pale yellow). It contains the primarily stalled, leading ribosome followed the colliding ribosome. For NGD RQC+ , cleavages occur on mRNA covered by the disome, whereby the leading ribosome undergoes RQC. In the mutant Hel2 lacking the C‐terminus, an alternative NGD pathway takes place (NGD RQC− ). Here, cleavages occur on mRNA covered by the ribosomes following the disome unit (blue) and the leading ribosome is not affected by RQC (grey).
    Figure Legend Snippet: Hel2‐mediated K63‐linked polyubiquitination is crucial for NGD and RQC Schematic drawing of the R(CGN) 12 reporter mRNA including the two quality control pathways induced by the R(CGN) 12 translation arrest sequence. Ribosome stalling occurs during translation of the R(CGN) 12 arrest sequence (shown in orange) and induces RQC and NGD. In the RQC pathway, the stalled ribosome is dissociated into subunits, and peptidyl‐tRNA remaining on the 60S subunit is ubiquitinated by Ltn1 (shown in pink) and degraded by the proteasome. In the NGD pathway, an endonucleolytic cleavage produces two fragments, the 5′ NGD intermediate (5′ NGD‐IM) and 3′ NGD intermediate (3′ NGD‐IM). The green and thin grey lines indicate GFP and HIS3 open reading frames (ORFs), and the black line indicates an untranslated region (UTR). Schematic drawing of the truncated Hel2 mutant proteins. Activities in RQC or NGD induced by the R(CGN) 12 sequence are indicated. Western blot showing that Hel2(1–315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN) 12 reporter in ltn1 Δ cells expressing truncated Hel2 mutant protein were detected with an anti‐GFP antibody. Northern blot showing the 5′ NGD‐IM derived from the R(CGN) 12 reporter in ski2 Δ cells expressing the indicated Hel2 mutant proteins. 5′ NGD‐IMs were detected with a DIG‐labelled GFP probe. Primer extension mapping of 5′ ends of 3′ NGD intermediates in Hel2‐WT or Hel2(1–315) mutant cells at nucleotide resolution. The primer extension samples were analysed using 5% TBE‐Urea‐PAGE and detected by fluorescence. Non‐specific reverse transcription (ReTr) products are indicated by asterisks. Dissection of NGD RQC+ and NGD RQC− : the carboxyl‐terminal region of Hel2 is required for both NGD and RQC which is likely triggered on a disome unit (pale yellow). It contains the primarily stalled, leading ribosome followed the colliding ribosome. For NGD RQC+ , cleavages occur on mRNA covered by the disome, whereby the leading ribosome undergoes RQC. In the mutant Hel2 lacking the C‐terminus, an alternative NGD pathway takes place (NGD RQC− ). Here, cleavages occur on mRNA covered by the ribosomes following the disome unit (blue) and the leading ribosome is not affected by RQC (grey).

    Techniques Used: Sequencing, Mutagenesis, Western Blot, Derivative Assay, Expressing, Northern Blot, Polyacrylamide Gel Electrophoresis, Fluorescence, Dissection

    Alteration of mRNA cleavage sites in Hel2(1–315) expressing cells A, B Northern blot showing that the length of 5′ NGD intermediate was altered in Hel2(1–315) expressing cells and slh1 ∆ cells. The full‐length GFP‐R(CGN) 12 ‐FLAG‐HIS3 mRNA and 5′ NGD intermediates (5′ NGD‐IM) or 3′ NGD intermediates (3′ NGD‐IM) were detected in the indicated mutant cells with expression of Hel2 wild‐type or 1–315 mutant from plasmid by Northern blotting with DIG‐labelled probes. 5′ NGD intermediates were detected by DIG‐labelled GFP probe in (A), and 3′ NGD intermediates were detected by the DIG‐labelled HIS3 probe in (B). SCR1 is used as loading control. FL; full length. Note an upstream shift of NGD cleavage sites in lanes A3‐4 and B3‐4.
    Figure Legend Snippet: Alteration of mRNA cleavage sites in Hel2(1–315) expressing cells A, B Northern blot showing that the length of 5′ NGD intermediate was altered in Hel2(1–315) expressing cells and slh1 ∆ cells. The full‐length GFP‐R(CGN) 12 ‐FLAG‐HIS3 mRNA and 5′ NGD intermediates (5′ NGD‐IM) or 3′ NGD intermediates (3′ NGD‐IM) were detected in the indicated mutant cells with expression of Hel2 wild‐type or 1–315 mutant from plasmid by Northern blotting with DIG‐labelled probes. 5′ NGD intermediates were detected by DIG‐labelled GFP probe in (A), and 3′ NGD intermediates were detected by the DIG‐labelled HIS3 probe in (B). SCR1 is used as loading control. FL; full length. Note an upstream shift of NGD cleavage sites in lanes A3‐4 and B3‐4.

    Techniques Used: Expressing, Northern Blot, Mutagenesis, Plasmid Preparation

    uS10 in colliding ribosome is efficiently ubiquitinated A Top: schematic drawing of the (CGA‐CCG) dicodon reporter mRNA used for in vitro translation experiments. Bottom: scheme outlining the principle of ribosome–nascent chain (RNC) purification. Stalled and colliding ribosomes are pulled down via an affinity tag on the nascent chain. B, C Both RQC and NGD RQC+ are triggered by a (CGA‐CCG) dicodon containing arrest sequence in vivo . (B) Western blot showing the arrest products derived from the GFP‐X‐FLAG‐HIS3 reporter. Translation products were detected by Western blot using an anti‐GFP antibody. (C) Northern blot for the 5′ NGD‐IM derived from the (CGA‐CCG) reporter in ski2 Δ cells. 5′ NGD‐IMs were detected with a DIG‐labelled GFP probe. SCR1 was used as a load control. D Western blot of test translations using the (CGA‐CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2 Δ uS10‐3HA strain. Expression of the translation products (free His‐ and V5‐tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti‐V5 antibody. E Sucrose gradient fractions (top) and Western blot analysis (bottom) of the (CGA‐CCG) dicodon‐stalled RNC samples. After the translation reaction, the RNCs were affinity‐purified as indicated in (C). The eluate was loaded on a 10–50% sucrose gradient and fractionated. Each collected fraction was analysed using anti‐HA antibody to detect uS10‐HA. Note that disomes are preferentially polyubiquitinated over monosomes.
    Figure Legend Snippet: uS10 in colliding ribosome is efficiently ubiquitinated A Top: schematic drawing of the (CGA‐CCG) dicodon reporter mRNA used for in vitro translation experiments. Bottom: scheme outlining the principle of ribosome–nascent chain (RNC) purification. Stalled and colliding ribosomes are pulled down via an affinity tag on the nascent chain. B, C Both RQC and NGD RQC+ are triggered by a (CGA‐CCG) dicodon containing arrest sequence in vivo . (B) Western blot showing the arrest products derived from the GFP‐X‐FLAG‐HIS3 reporter. Translation products were detected by Western blot using an anti‐GFP antibody. (C) Northern blot for the 5′ NGD‐IM derived from the (CGA‐CCG) reporter in ski2 Δ cells. 5′ NGD‐IMs were detected with a DIG‐labelled GFP probe. SCR1 was used as a load control. D Western blot of test translations using the (CGA‐CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2 Δ uS10‐3HA strain. Expression of the translation products (free His‐ and V5‐tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti‐V5 antibody. E Sucrose gradient fractions (top) and Western blot analysis (bottom) of the (CGA‐CCG) dicodon‐stalled RNC samples. After the translation reaction, the RNCs were affinity‐purified as indicated in (C). The eluate was loaded on a 10–50% sucrose gradient and fractionated. Each collected fraction was analysed using anti‐HA antibody to detect uS10‐HA. Note that disomes are preferentially polyubiquitinated over monosomes.

    Techniques Used: In Vitro, Purification, Sequencing, In Vivo, Western Blot, Derivative Assay, Northern Blot, Expressing, Affinity Purification

    36) Product Images from "Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules"

    Article Title: Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.283

    SNX-BAR proteins show a specific pattern of homo- and hetero-dimerization. ( A ) Western blots (WBs) of expressed Flag–SNX4, Flag–SNX7 or Flag–SNX30 co-expressing GST–SNX1 to GST–SNX9, GST–SNX30, GST–SNX32 or GST–SNX33 in HEK-293T cells using glutathione-sepharose precipitation (GST-P). ( B ) WBs of expressed Flag–SNX8 co-expressing GFP–SNX1, GFP–SNX4, GFP–SNX5, GFP–SNX8, GFP–SNX9, GFP–SNX18 or GFP–SNX33 in HEK-293T cells using GFP-nanotrap immunoprecipitation (IP). ( C ) WBs of expressed Flag–SNX9, mCherry–SNX18 or Flag–SNX33 co-expressing GFP–SNX1, GFP–SNX4, GFP–SNX5, GFP–SNX8, GFP–SNX9, GFP–SNX18 or GFP–SNX33 in HEK-293T cells using GFP-nanotrap IP. Figure source data can be found with the Supplementary data .
    Figure Legend Snippet: SNX-BAR proteins show a specific pattern of homo- and hetero-dimerization. ( A ) Western blots (WBs) of expressed Flag–SNX4, Flag–SNX7 or Flag–SNX30 co-expressing GST–SNX1 to GST–SNX9, GST–SNX30, GST–SNX32 or GST–SNX33 in HEK-293T cells using glutathione-sepharose precipitation (GST-P). ( B ) WBs of expressed Flag–SNX8 co-expressing GFP–SNX1, GFP–SNX4, GFP–SNX5, GFP–SNX8, GFP–SNX9, GFP–SNX18 or GFP–SNX33 in HEK-293T cells using GFP-nanotrap immunoprecipitation (IP). ( C ) WBs of expressed Flag–SNX9, mCherry–SNX18 or Flag–SNX33 co-expressing GFP–SNX1, GFP–SNX4, GFP–SNX5, GFP–SNX8, GFP–SNX9, GFP–SNX18 or GFP–SNX33 in HEK-293T cells using GFP-nanotrap IP. Figure source data can be found with the Supplementary data .

    Techniques Used: Western Blot, Expressing, Immunoprecipitation

    Charged residues in the hydrophobic BAR interface determine specific dimerization of SNX-BAR proteins. ( A ) The homology model of the SNX5:SNX5 homodimer (monomers shown in blue and cyan ribbons). Clashing negatively charged Glu280 and Glu383 are shown as red space filling spheres. ( B ) Close-up of the environment of Glu280 and Glu383 in the SNX5:SNX5 dimerization interface. ( C ) Western blots (WB) of expressed Flag–SNX5 wild type (WT) or Flag–SNX5-E280A/E383A (AA) co-expressing GFP control, GFP–SNX1, GFP–SNX5-WT or GFP–SNX5-AA in HEK-293T cells using GFP-nanotrap IP. ( D ) Example micrographs of liposomes incubated with 10 μM SNX5-AA (i–iii show three different example views), scale bar represents 200 nm. ( E ) Coomassie-stained gel of SNX5-AA in the pellet (P) and supernatant (S) fractions after sedimentation in the presence or absence of liposomes. Figure source data can be found with the Supplementary data .
    Figure Legend Snippet: Charged residues in the hydrophobic BAR interface determine specific dimerization of SNX-BAR proteins. ( A ) The homology model of the SNX5:SNX5 homodimer (monomers shown in blue and cyan ribbons). Clashing negatively charged Glu280 and Glu383 are shown as red space filling spheres. ( B ) Close-up of the environment of Glu280 and Glu383 in the SNX5:SNX5 dimerization interface. ( C ) Western blots (WB) of expressed Flag–SNX5 wild type (WT) or Flag–SNX5-E280A/E383A (AA) co-expressing GFP control, GFP–SNX1, GFP–SNX5-WT or GFP–SNX5-AA in HEK-293T cells using GFP-nanotrap IP. ( D ) Example micrographs of liposomes incubated with 10 μM SNX5-AA (i–iii show three different example views), scale bar represents 200 nm. ( E ) Coomassie-stained gel of SNX5-AA in the pellet (P) and supernatant (S) fractions after sedimentation in the presence or absence of liposomes. Figure source data can be found with the Supplementary data .

    Techniques Used: Western Blot, Expressing, Incubation, Staining, Sedimentation

    VPS5 homologues of the SNX-BAR-retromer complex remodel liposomes into tubular membrane structures. ( A ) Example micrographs of negative stained liposomes, extruded to 200 nm diameter and incubated with buffer control or the individual SNX-BAR proteins of the mammalian SNX-BAR-retromer complex at 10 μM final concentration (i–iii show three different example views). ( B ) Example micrographs of liposomes incubated with Trypanosoma brucei VPS5 (TbVPS5), Saccharomyces cerevisiae VPS5 (ScVPS5) or Caenorhabditis elegans SNX1 (CeSNX1) and SNX6 (CeSNX6) at 10 μM final concentration (i–iii show three different example views). ( C ) Example micrographs of liposomes incubated with the trimeric complex of Homo sapiens VPS26–VPS29–VPS36 (i–iii show three different example views). ( D ) Confocal image of a HeLa cell expressing GFP–SNX1 (green) and stained for nucleus (DAPI, blue). The boxed area indicates the region that is displayed in the insert. Arrowheads indicate GFP–SNX1-positive tubular structures. Scale bar represents 10 μm. ( E ) Electron micrograph of an endosome (marked by ‘E') in a HeLa cell expressing GFP–SNX1, which is processed according to the Tokuyasu cryosection method and immunolabelled for GFP-10 nm gold. Arrowheads indicate the tubular/vesicular profiles positive for GFP–SNX1. ( F ) Electron micrograph of liposomes incubated with 10 μM SNX1 at similar magnification as ( E ). ( G ) Histogram of the minimal diameter of SNX1-positive tubular/vesicular profiles in Tokuyasu-processed HeLa cells ( n =105) and SNX1-formed tubules on 200 nm liposomes in vitro ( n =49), plotted as percentage of all tubules in 5 nm bins. All arrowheads indicate the membrane tubules. All scale bars represent 200 nm unless otherwise indicated.
    Figure Legend Snippet: VPS5 homologues of the SNX-BAR-retromer complex remodel liposomes into tubular membrane structures. ( A ) Example micrographs of negative stained liposomes, extruded to 200 nm diameter and incubated with buffer control or the individual SNX-BAR proteins of the mammalian SNX-BAR-retromer complex at 10 μM final concentration (i–iii show three different example views). ( B ) Example micrographs of liposomes incubated with Trypanosoma brucei VPS5 (TbVPS5), Saccharomyces cerevisiae VPS5 (ScVPS5) or Caenorhabditis elegans SNX1 (CeSNX1) and SNX6 (CeSNX6) at 10 μM final concentration (i–iii show three different example views). ( C ) Example micrographs of liposomes incubated with the trimeric complex of Homo sapiens VPS26–VPS29–VPS36 (i–iii show three different example views). ( D ) Confocal image of a HeLa cell expressing GFP–SNX1 (green) and stained for nucleus (DAPI, blue). The boxed area indicates the region that is displayed in the insert. Arrowheads indicate GFP–SNX1-positive tubular structures. Scale bar represents 10 μm. ( E ) Electron micrograph of an endosome (marked by ‘E') in a HeLa cell expressing GFP–SNX1, which is processed according to the Tokuyasu cryosection method and immunolabelled for GFP-10 nm gold. Arrowheads indicate the tubular/vesicular profiles positive for GFP–SNX1. ( F ) Electron micrograph of liposomes incubated with 10 μM SNX1 at similar magnification as ( E ). ( G ) Histogram of the minimal diameter of SNX1-positive tubular/vesicular profiles in Tokuyasu-processed HeLa cells ( n =105) and SNX1-formed tubules on 200 nm liposomes in vitro ( n =49), plotted as percentage of all tubules in 5 nm bins. All arrowheads indicate the membrane tubules. All scale bars represent 200 nm unless otherwise indicated.

    Techniques Used: Staining, Incubation, Concentration Assay, Expressing, In Vitro

    37) Product Images from "NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope"

    Article Title: NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111851

    The NET23/STING chromatin compaction effect does not depend on H2B-GFP or the epitope tag used and occurs in a wide range of cell types. ( A ) Though at later times (72 h post-transfection) the compacted chromatin in the H2B-GFP HeLa cells was distributed throughout the nucleus ( Figure 1 ), at 21 h post-transfection a large percentage of the compacted chromatin could be observed at the nuclear periphery. In this case, the compaction shown was visualized using DAPI to stain the DNA that yielded similar changes as observed for the H2B-GFP signal, indicating that outputs in subsequent experiments using other cell lines without the H2B-GFP could be compared. ( B ) The effect of NET23/STING is independent of the epitope tag used. NET23/STING with a large C-terminal mRFP tag (upper panels) or a small N-terminal HA tag (lower panels) both yielded the chromatin compaction phenotype in the H2B-GFP HeLa cells, again using DAPI staining to visualize the DNA. The NET is shown in red and the DAPI staining for DNA in grey. ( C ) The chromatin compaction phenotype of NET23/STING was not cell type dependent as the effect could be observed in MRC5 primary human lung fibroblasts, 216−/− lamin A knockout mouse embryonic fibroblasts, U2OS human osteocarcinoma cells, HepG2 human liver cancer cells, HEK/293T human embryonic kidney cells, and NIH3T3 mouse fibroblasts. Again, the NET23/STING is shown in red and the DAPI staining for DNA in grey. All scale bars = 10 µm.
    Figure Legend Snippet: The NET23/STING chromatin compaction effect does not depend on H2B-GFP or the epitope tag used and occurs in a wide range of cell types. ( A ) Though at later times (72 h post-transfection) the compacted chromatin in the H2B-GFP HeLa cells was distributed throughout the nucleus ( Figure 1 ), at 21 h post-transfection a large percentage of the compacted chromatin could be observed at the nuclear periphery. In this case, the compaction shown was visualized using DAPI to stain the DNA that yielded similar changes as observed for the H2B-GFP signal, indicating that outputs in subsequent experiments using other cell lines without the H2B-GFP could be compared. ( B ) The effect of NET23/STING is independent of the epitope tag used. NET23/STING with a large C-terminal mRFP tag (upper panels) or a small N-terminal HA tag (lower panels) both yielded the chromatin compaction phenotype in the H2B-GFP HeLa cells, again using DAPI staining to visualize the DNA. The NET is shown in red and the DAPI staining for DNA in grey. ( C ) The chromatin compaction phenotype of NET23/STING was not cell type dependent as the effect could be observed in MRC5 primary human lung fibroblasts, 216−/− lamin A knockout mouse embryonic fibroblasts, U2OS human osteocarcinoma cells, HepG2 human liver cancer cells, HEK/293T human embryonic kidney cells, and NIH3T3 mouse fibroblasts. Again, the NET23/STING is shown in red and the DAPI staining for DNA in grey. All scale bars = 10 µm.

    Techniques Used: Transfection, Staining, Knock-Out

    A screen for NETs that alter chromatin compaction. ( A ) 72 h post-transfection HeLa cells have no gross changes in distribution of H2B-GFP (green) when most NETs fused to mRFP (red) are exogenously expressed (e.g. emerin and NET51, upper panels). However, cells transfected with NET23/STING (lower panels) exhibit considerable chromatin compaction. ( B ) Zoomed images of chromatin in untransfected (left) and NET23 transfected cells. All images were taken using identical settings and all scale bars = 10 µm.
    Figure Legend Snippet: A screen for NETs that alter chromatin compaction. ( A ) 72 h post-transfection HeLa cells have no gross changes in distribution of H2B-GFP (green) when most NETs fused to mRFP (red) are exogenously expressed (e.g. emerin and NET51, upper panels). However, cells transfected with NET23/STING (lower panels) exhibit considerable chromatin compaction. ( B ) Zoomed images of chromatin in untransfected (left) and NET23 transfected cells. All images were taken using identical settings and all scale bars = 10 µm.

    Techniques Used: Transfection

    NET23/STING promotes apoptosis. ( A ) Gating strategy used for cells in B using forward and side scatter profiles to exclude debris followed by DNA content to determine intact singlet cells. The transfected population (expressing GFP) was identified by subsequently gating singlet cellular material on forward scatter versus GFP intensity. All cells in this experiment were analyzed at 44 h post-transfection. ( B ) The cells used to determine the gates were also stained for propidium iodide (PI; y-axis) and annexin V (x-axis). The traces in the left panels show the untransfected cells in the population and those in the right panels show the cells with GFP signal. The right-most green peak delineates cells with an annexin V signal of sufficient intensity to indicate cells undergoing apoptosis. As expected, for the mock-transfected culture essentially no GFP positive cells were identified and very few apoptosing cells could be observed. Expression of NET23/STING consistently increased the apoptosing population regardless of whether the tag was on the N-terminus (GFP-NET23) or the C-terminus (NET23-GFP) and the effect of NET23/STING did not require function of the master regulator p53 as apoptosis was induced in both wild-type (p53 +/+ ) and p53 knockout (p53 −/− ) cells. Nonetheless, it is notable that the responses were very similar between the two NET23/STING constructs in the wild-type cells while the N-terminal tag showed a lagging apoptotic response in the p53 knockout cells. ( C ) The percentage of annexin V-positive cells is plotted after correction to subtract the number in the GFP control with the wild-type (p53 +/+ ) cells. This is used as the correction for both cell lines to better indicate the effect of the p53 knockout itself on apoptosis induction.
    Figure Legend Snippet: NET23/STING promotes apoptosis. ( A ) Gating strategy used for cells in B using forward and side scatter profiles to exclude debris followed by DNA content to determine intact singlet cells. The transfected population (expressing GFP) was identified by subsequently gating singlet cellular material on forward scatter versus GFP intensity. All cells in this experiment were analyzed at 44 h post-transfection. ( B ) The cells used to determine the gates were also stained for propidium iodide (PI; y-axis) and annexin V (x-axis). The traces in the left panels show the untransfected cells in the population and those in the right panels show the cells with GFP signal. The right-most green peak delineates cells with an annexin V signal of sufficient intensity to indicate cells undergoing apoptosis. As expected, for the mock-transfected culture essentially no GFP positive cells were identified and very few apoptosing cells could be observed. Expression of NET23/STING consistently increased the apoptosing population regardless of whether the tag was on the N-terminus (GFP-NET23) or the C-terminus (NET23-GFP) and the effect of NET23/STING did not require function of the master regulator p53 as apoptosis was induced in both wild-type (p53 +/+ ) and p53 knockout (p53 −/− ) cells. Nonetheless, it is notable that the responses were very similar between the two NET23/STING constructs in the wild-type cells while the N-terminal tag showed a lagging apoptotic response in the p53 knockout cells. ( C ) The percentage of annexin V-positive cells is plotted after correction to subtract the number in the GFP control with the wild-type (p53 +/+ ) cells. This is used as the correction for both cell lines to better indicate the effect of the p53 knockout itself on apoptosis induction.

    Techniques Used: Transfection, Expressing, Staining, Knock-Out, Construct

    NET23/STING chromatin effects may set the stage for a transitional state between chromatin condensation and apoptosis. ( A ) Cells were taken at 23 h post-transfection and stained for DNA and the characteristic early apoptosis marker annexin V. GFP-transfected cells exhibit a normal distribution pattern with a large annexin V-negative G1 population (close to 100K) and smaller annexin V-negative G2/M population (close to 200K) and a small (∼10%) sub-G1 population that is mostly annexin V-positive. In contrast, at this early time post-transfection the NET23/STING-transfected population yields an aberrant distribution pattern with the main cell populations slightly lower than the normal G1 population, yet still slightly larger than the apoptosing sub-G1 population. This may reflect the process of chromatin condensation. ( B ) To investigate this population further, NET23/STING-transfected cells were analyzed over a timecourse from 17 to 66 h post-transfection. Over time the higher sub-G1 population can be observed to initially increase and then diminish as the smaller sub-G1 population increases. The density plots shown on the left plot DNA content against forward scatter to measure overall cell size/shape and thus likely give information about the shift in chromatin compaction, but these plots can be misleading about total numbers because of spots representing individual cells being printed on top of one another. In contrast, the cell cycle population plots on the right clearly show the total percentage of cells for the initial appearance of a higher sub-G1 population followed by its chasing into an apoptotic smaller/fragmented sub-G1 population.
    Figure Legend Snippet: NET23/STING chromatin effects may set the stage for a transitional state between chromatin condensation and apoptosis. ( A ) Cells were taken at 23 h post-transfection and stained for DNA and the characteristic early apoptosis marker annexin V. GFP-transfected cells exhibit a normal distribution pattern with a large annexin V-negative G1 population (close to 100K) and smaller annexin V-negative G2/M population (close to 200K) and a small (∼10%) sub-G1 population that is mostly annexin V-positive. In contrast, at this early time post-transfection the NET23/STING-transfected population yields an aberrant distribution pattern with the main cell populations slightly lower than the normal G1 population, yet still slightly larger than the apoptosing sub-G1 population. This may reflect the process of chromatin condensation. ( B ) To investigate this population further, NET23/STING-transfected cells were analyzed over a timecourse from 17 to 66 h post-transfection. Over time the higher sub-G1 population can be observed to initially increase and then diminish as the smaller sub-G1 population increases. The density plots shown on the left plot DNA content against forward scatter to measure overall cell size/shape and thus likely give information about the shift in chromatin compaction, but these plots can be misleading about total numbers because of spots representing individual cells being printed on top of one another. In contrast, the cell cycle population plots on the right clearly show the total percentage of cells for the initial appearance of a higher sub-G1 population followed by its chasing into an apoptotic smaller/fragmented sub-G1 population.

    Techniques Used: Transfection, Staining, Marker

    NET23/STING chromatin effects are independent of apoptosis and result in an increase in G2/M. ( A ) Untransfected cells (Mock), NET23-GFP transfected cells, and NET23-GFP transfected HT1080 cells treated with 20 µM of the pan-caspase inhibitor Z-VAD were stained for DNA content with the permeable dye Hoechst 33342 and the characteristic early apoptosis marker annexin V. The total sub-G1 population is gated (pink box) and anything above roughly 10 3 should be positive for annexin V. Both the lower sub-G1 population and most of the annexin V staining of the NET23-GFP population are absent from the Z-VAD treated population. ( B ) HT1080 cells were similarly treated, fixed and stained for DNA for microscopy. Despite the blocking of apoptosis pathways with the pan-caspase inhibitor, the chromatin compaction still occurred in the NET23/STING transfected cells. Scale bars = 10 µm.
    Figure Legend Snippet: NET23/STING chromatin effects are independent of apoptosis and result in an increase in G2/M. ( A ) Untransfected cells (Mock), NET23-GFP transfected cells, and NET23-GFP transfected HT1080 cells treated with 20 µM of the pan-caspase inhibitor Z-VAD were stained for DNA content with the permeable dye Hoechst 33342 and the characteristic early apoptosis marker annexin V. The total sub-G1 population is gated (pink box) and anything above roughly 10 3 should be positive for annexin V. Both the lower sub-G1 population and most of the annexin V staining of the NET23-GFP population are absent from the Z-VAD treated population. ( B ) HT1080 cells were similarly treated, fixed and stained for DNA for microscopy. Despite the blocking of apoptosis pathways with the pan-caspase inhibitor, the chromatin compaction still occurred in the NET23/STING transfected cells. Scale bars = 10 µm.

    Techniques Used: Transfection, Staining, Marker, Microscopy, Blocking Assay

    38) Product Images from "SLEEPLESS, a Ly-6/Neurotoxin Family Member, Regulates Levels, Localization, and Activity of Shaker"

    Article Title: SLEEPLESS, a Ly-6/Neurotoxin Family Member, Regulates Levels, Localization, and Activity of Shaker

    Journal: Nature neuroscience

    doi: 10.1038/nn.2454

    Rescue of Shaker expression in sss mutants by transgenic expression of sss ( a – d ) OK107–Gal4 was used to direct expression of GFP ( a , c ) or sss ( b , d ), and GFP or Shaker expression was examined, respectively. Since Shaker was enriched in synapse–rich neuropil, GFP fused to synaptogamin ( syt –GFP), which is targeted to the synapse, was used. GFP expression was seen in the mushroom bodies ( a ), but not in the group of visual projection neurons (VPNs) sending processes to the optic lobe ( c ). Transgenic expression of sss using OK107–Gal4 increased Shaker expression in the mushroom bodies ( b ), but not in the optic lobe ( d ). ( e – h ) vGlut –Gal4 directed GFP expression in the visual projection neurons and the optic lobe ( g ), but not the mushroom bodies ( e ). Transgenic expression of sss using vGlut –Gal4 increased Shaker expression in the optic lobe ( h ), but not in the mushroom bodies ( f ). Arrows point to the fiber bundles formed by the VPNs. Maximal intensity projections of seven 1–μm sections from the anterior of the brain are shown for ( a ), ( b ), ( e ), and ( f ), and a single 1–μm section from the posterior of the brain at the level of the protocerebral bridge is shown for ( c ), ( d ), ( g ), and ( h ). Representative brains are shown, taken from two independent experiments. n=5 or 6 for all genotypes. Scale bar in ( a ) for ( a , b , e , f ) and ( c ) for ( c , d , g , h ), 50 μm
    Figure Legend Snippet: Rescue of Shaker expression in sss mutants by transgenic expression of sss ( a – d ) OK107–Gal4 was used to direct expression of GFP ( a , c ) or sss ( b , d ), and GFP or Shaker expression was examined, respectively. Since Shaker was enriched in synapse–rich neuropil, GFP fused to synaptogamin ( syt –GFP), which is targeted to the synapse, was used. GFP expression was seen in the mushroom bodies ( a ), but not in the group of visual projection neurons (VPNs) sending processes to the optic lobe ( c ). Transgenic expression of sss using OK107–Gal4 increased Shaker expression in the mushroom bodies ( b ), but not in the optic lobe ( d ). ( e – h ) vGlut –Gal4 directed GFP expression in the visual projection neurons and the optic lobe ( g ), but not the mushroom bodies ( e ). Transgenic expression of sss using vGlut –Gal4 increased Shaker expression in the optic lobe ( h ), but not in the mushroom bodies ( f ). Arrows point to the fiber bundles formed by the VPNs. Maximal intensity projections of seven 1–μm sections from the anterior of the brain are shown for ( a ), ( b ), ( e ), and ( f ), and a single 1–μm section from the posterior of the brain at the level of the protocerebral bridge is shown for ( c ), ( d ), ( g ), and ( h ). Representative brains are shown, taken from two independent experiments. n=5 or 6 for all genotypes. Scale bar in ( a ) for ( a , b , e , f ) and ( c ) for ( c , d , g , h ), 50 μm

    Techniques Used: Expressing, Transgenic Assay

    39) Product Images from "Antibodies on demand: a fast method for the production of human scFvs with minimal amounts of antigen"

    Article Title: Antibodies on demand: a fast method for the production of human scFvs with minimal amounts of antigen

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-61

    Printing conditions and scFv antibody microarrays reproducibility . A) Printing map of a microarray comprising 384 scFvs against GFP or Trx. Controls used in the assay were: red box, mAb anti-T7Tag, 1:10 diluted. Yellow box, mAb anti-T7Tag 1:100 diluted. Blue box, crude Trx (top) or GFP (bottom) RTS extract 1:10 diluted. Grey box, printing buffer. Green box, TA4 anti-gastrin17 scFv; from right to left 1:10, 1:100, 1:1000 and 1:1000 dilutions. B) A representative image of a microarray probed with an anti-c-myc antibody to assess the correct printing of the scFvs. For detecting c-myc antibody, slides were incubated with Alexa Fluor 555-labeled goat anti-mouse IgG antibodies. White spots indicate a saturation of the green signal intensity. C) scFvs were spotted in duplicate onto FAST nitrocellulose coated slides to verify the intra-assay reproducibility. Replicated spots showed a uniform intensity either visually or by GenePix analysis. The two intensity values for each clone were quantified and plotted to assess the intra-array reproducibility.
    Figure Legend Snippet: Printing conditions and scFv antibody microarrays reproducibility . A) Printing map of a microarray comprising 384 scFvs against GFP or Trx. Controls used in the assay were: red box, mAb anti-T7Tag, 1:10 diluted. Yellow box, mAb anti-T7Tag 1:100 diluted. Blue box, crude Trx (top) or GFP (bottom) RTS extract 1:10 diluted. Grey box, printing buffer. Green box, TA4 anti-gastrin17 scFv; from right to left 1:10, 1:100, 1:1000 and 1:1000 dilutions. B) A representative image of a microarray probed with an anti-c-myc antibody to assess the correct printing of the scFvs. For detecting c-myc antibody, slides were incubated with Alexa Fluor 555-labeled goat anti-mouse IgG antibodies. White spots indicate a saturation of the green signal intensity. C) scFvs were spotted in duplicate onto FAST nitrocellulose coated slides to verify the intra-assay reproducibility. Replicated spots showed a uniform intensity either visually or by GenePix analysis. The two intensity values for each clone were quantified and plotted to assess the intra-array reproducibility.

    Techniques Used: Microarray, Incubation, Labeling, Intra Assay

    Cell-free protein expression and characterization . A) Cell free GFP and Trx expression and purification by TALON™ Dynabeads ® was assessed by SDS-PAGE and Coomassie Blue staining. U, unpurified total protein extract. P, protein purified with TALON™ Dynabeads ® . B) Western blot analysis of the proteins by using peroxidase-labeled anti-His. U, unpurified total protein extract. P, Proteins purified with TALON™ Dynabeads ® . C) ELISA characterization of the polyclonal phages after different rounds of biopanning. A peroxidase-labeled anti-M13 was followed by TMB incubation to develop the signal. His-tagged BSA and GST were used as negative controls.
    Figure Legend Snippet: Cell-free protein expression and characterization . A) Cell free GFP and Trx expression and purification by TALON™ Dynabeads ® was assessed by SDS-PAGE and Coomassie Blue staining. U, unpurified total protein extract. P, protein purified with TALON™ Dynabeads ® . B) Western blot analysis of the proteins by using peroxidase-labeled anti-His. U, unpurified total protein extract. P, Proteins purified with TALON™ Dynabeads ® . C) ELISA characterization of the polyclonal phages after different rounds of biopanning. A peroxidase-labeled anti-M13 was followed by TMB incubation to develop the signal. His-tagged BSA and GST were used as negative controls.

    Techniques Used: Expressing, Purification, SDS Page, Staining, Western Blot, Labeling, Enzyme-linked Immunosorbent Assay, Incubation

    Screening of specific scFvs using a microarray format . Cell free-expressed proteins were labeled with 647 AlexaFluor and incubated with GFP- and Trx-specific scFvs microarrays to identify highly specific scFv binders. E. coli- expressed proteins were used as a control. A) Selection of GFP-specific scFvs. Left, performance of the microarray with cell free-expressed 647-labeled GFP or Trx as control. Right, performance of the microarray with E. coli expressed proteins followed by a polyclonal anti-GFP or a monoclonal anti-Flag and by AlexaFluor 555 labeled antibodies gave a green fluorescent signal. White boxes: 10 scFv antibodies against GFP showing at least 3-fold higher fluorescence signal than the control Trx values. Red boxes: scFvs showing non-specific binding for GFP. B) Anti-GFP scFv clones were tested by ELISA using GFP and GST to compare the microarray technology with ELISA screening. C) Selection of Trx-specific scFvs. Left, performance of the microarray with cell free-expressed 647-labeled Trx or GFP as control. Right, performance of the microarray with E. coli -expressed proteins followed by a polyclonal anti-GFP antibody or a monoclonal anti-Flag and by AlexaFluor 555 labeled antibodies. White boxes: 8 reactive scFv antibodies detected by antibody microarrays against Trx showing at least 3-fold higher fluorescence signal than the control GFP values. Red boxes: scFvs showing non-specific binding for Trx. D) Anti-Trx scFv clones were tested by ELISA using Trx, GST-His6 tagged and BSA to compare the performance of the microarray technology to identify Trx scFv binders with ELISA screening. Green arrows indicate the top three scFvs that gave the highest microarray signal for GFP or Trx and were further validated by other immunological techniques.
    Figure Legend Snippet: Screening of specific scFvs using a microarray format . Cell free-expressed proteins were labeled with 647 AlexaFluor and incubated with GFP- and Trx-specific scFvs microarrays to identify highly specific scFv binders. E. coli- expressed proteins were used as a control. A) Selection of GFP-specific scFvs. Left, performance of the microarray with cell free-expressed 647-labeled GFP or Trx as control. Right, performance of the microarray with E. coli expressed proteins followed by a polyclonal anti-GFP or a monoclonal anti-Flag and by AlexaFluor 555 labeled antibodies gave a green fluorescent signal. White boxes: 10 scFv antibodies against GFP showing at least 3-fold higher fluorescence signal than the control Trx values. Red boxes: scFvs showing non-specific binding for GFP. B) Anti-GFP scFv clones were tested by ELISA using GFP and GST to compare the microarray technology with ELISA screening. C) Selection of Trx-specific scFvs. Left, performance of the microarray with cell free-expressed 647-labeled Trx or GFP as control. Right, performance of the microarray with E. coli -expressed proteins followed by a polyclonal anti-GFP antibody or a monoclonal anti-Flag and by AlexaFluor 555 labeled antibodies. White boxes: 8 reactive scFv antibodies detected by antibody microarrays against Trx showing at least 3-fold higher fluorescence signal than the control GFP values. Red boxes: scFvs showing non-specific binding for Trx. D) Anti-Trx scFv clones were tested by ELISA using Trx, GST-His6 tagged and BSA to compare the performance of the microarray technology to identify Trx scFv binders with ELISA screening. Green arrows indicate the top three scFvs that gave the highest microarray signal for GFP or Trx and were further validated by other immunological techniques.

    Techniques Used: Microarray, Labeling, Incubation, Selection, Fluorescence, Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay

    Application of the scFvs in ELISA and WB . A) Selected scFvs were tested by ELISA against the cell free-expressed proteins, E. coli- derived GFP and scFv-Trx 3xFlag proteins, using BSA and GST as negative controls. B) Intensity of the fluorescence signal of the GFP or Trx-specific scFvs in comparison to a scFv control signal or buffer obtained from the normalized data of the microarrays after incubation with 647Alexa Fluor labelled-GFP, 647Alexa Fluor labelled-Trx or anti c-myc followed by 555 AlexaFluor anti-mouse IgG, respectively. C) Different amounts of GFP or scFv-Trx were separated by SDS-PAGE and transferred to nitrocellulose membranes to determine the sensitivity of the anti-GFP and the anti-Trx scFvs (1:10 diluted) as primary antibodies. They were followed by an anti-c-myc tag and peroxidase-labeled anti-mouse IgG antibody, respectively. An anti-His6 mAb or an anti-M2 Flag mAb were used as positive controls to detect the target proteins.
    Figure Legend Snippet: Application of the scFvs in ELISA and WB . A) Selected scFvs were tested by ELISA against the cell free-expressed proteins, E. coli- derived GFP and scFv-Trx 3xFlag proteins, using BSA and GST as negative controls. B) Intensity of the fluorescence signal of the GFP or Trx-specific scFvs in comparison to a scFv control signal or buffer obtained from the normalized data of the microarrays after incubation with 647Alexa Fluor labelled-GFP, 647Alexa Fluor labelled-Trx or anti c-myc followed by 555 AlexaFluor anti-mouse IgG, respectively. C) Different amounts of GFP or scFv-Trx were separated by SDS-PAGE and transferred to nitrocellulose membranes to determine the sensitivity of the anti-GFP and the anti-Trx scFvs (1:10 diluted) as primary antibodies. They were followed by an anti-c-myc tag and peroxidase-labeled anti-mouse IgG antibody, respectively. An anti-His6 mAb or an anti-M2 Flag mAb were used as positive controls to detect the target proteins.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Fluorescence, Incubation, SDS Page, Labeling

    40) Product Images from "The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni"

    Article Title: The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni

    Journal: Nature Communications

    doi: 10.1038/ncomms11667

    RIP-seq analysis of C. jejuni CsrA. ( a ) Western blot analysis of coIP samples of C. jejuni NCTC11168 WT and csrA- 3xFLAG strains using anti-FLAG antibody confirms a successful CsrA-3xFLAG pulldown in the tagged strain. The amount of samples loaded (OD 600 of bacteria) is indicated. GroEL served as loading control. ( b ) Pie charts showing relative proportions of mapped cDNA reads of different RNA classes in the coIP libraries (hkRNA: housekeeping RNAs). Numbers in brackets indicate the relative enrichment of the respective RNA class in the CsrA-3xFLAG versus control coIPs. ( c ) Pie chart showing the percentages and enriched genes of mapped reads for all > 5-fold enriched CsrA target genes. ( d ) (Left) Mapped RNA-seq reads for the control (black) and CsrA-3xFLAG coIP (blue) in strain NCTC11168. Grey arrows: ORFs; black arrows: transcriptional start sites (TSS). Examples of enrichment patterns in 5′ UTRs ( flaA ) and between genes in a polycistron (Cj0310c-Cj0309c operon; encoding two paralogous efflux proteins). (Right) Western blot analysis using anti-FLAG and anti-GFP antibodies of reporter fusions to potential C. jejuni CsrA target genes in E. coli Δ pgaA , Δ pgaA /Δ csrA and Δ pgaA /Δ csrA +pBAD- csrA Cj (complementation with C. jejuni CsrA-Strep under control of an arabinose-inducible pBAD promoter) strains. Putative CsrA targets from C. jejuni were fused in-frame (for example, 33 aa for flaA ) to GFP or a FLAG- lacZ' tag ( Supplementary Fig. 4b ). As deletion of csrA dramatically enhanced biofilm formation and led to poor growth in liquid culture in our E. coli strain, reporter experiments were performed in a Δ pgaA background. GroEL served as loading control. Protein samples corresponding to 0.1 OD 600 were loaded. Quantifications of reporter expression are given below the blots. ( e ) (Left) CsrA-binding motif predicted by MEME 24 ( E -value=2.1E-11). (Right) Consensus secondary structure motif of C. jejuni CsrA-binding sites predicted by CMfinder 62 .
    Figure Legend Snippet: RIP-seq analysis of C. jejuni CsrA. ( a ) Western blot analysis of coIP samples of C. jejuni NCTC11168 WT and csrA- 3xFLAG strains using anti-FLAG antibody confirms a successful CsrA-3xFLAG pulldown in the tagged strain. The amount of samples loaded (OD 600 of bacteria) is indicated. GroEL served as loading control. ( b ) Pie charts showing relative proportions of mapped cDNA reads of different RNA classes in the coIP libraries (hkRNA: housekeeping RNAs). Numbers in brackets indicate the relative enrichment of the respective RNA class in the CsrA-3xFLAG versus control coIPs. ( c ) Pie chart showing the percentages and enriched genes of mapped reads for all > 5-fold enriched CsrA target genes. ( d ) (Left) Mapped RNA-seq reads for the control (black) and CsrA-3xFLAG coIP (blue) in strain NCTC11168. Grey arrows: ORFs; black arrows: transcriptional start sites (TSS). Examples of enrichment patterns in 5′ UTRs ( flaA ) and between genes in a polycistron (Cj0310c-Cj0309c operon; encoding two paralogous efflux proteins). (Right) Western blot analysis using anti-FLAG and anti-GFP antibodies of reporter fusions to potential C. jejuni CsrA target genes in E. coli Δ pgaA , Δ pgaA /Δ csrA and Δ pgaA /Δ csrA +pBAD- csrA Cj (complementation with C. jejuni CsrA-Strep under control of an arabinose-inducible pBAD promoter) strains. Putative CsrA targets from C. jejuni were fused in-frame (for example, 33 aa for flaA ) to GFP or a FLAG- lacZ' tag ( Supplementary Fig. 4b ). As deletion of csrA dramatically enhanced biofilm formation and led to poor growth in liquid culture in our E. coli strain, reporter experiments were performed in a Δ pgaA background. GroEL served as loading control. Protein samples corresponding to 0.1 OD 600 were loaded. Quantifications of reporter expression are given below the blots. ( e ) (Left) CsrA-binding motif predicted by MEME 24 ( E -value=2.1E-11). (Right) Consensus secondary structure motif of C. jejuni CsrA-binding sites predicted by CMfinder 62 .

    Techniques Used: Western Blot, Co-Immunoprecipitation Assay, RNA Sequencing Assay, Expressing, Binding Assay

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    In Vitro:

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    Roche adult spata7 gfp transgenic
    <t>SPATA7</t> extends AHI1 localization apical of the TZ in primary cilia. (a) Endogenous AHI1 (white) localizes at the TZ of the primary cilium, marked by acetylated α-tubulin in hTERT-RPE1 cells (left). Ectopic expression of <t>hSPATA7-GFP</t> (right) induces apical extension of the endogenous AHI1 localization signal beyond the TZ into the ciliary axoneme (subsets display zoomed images of AHI1 [white], acetylated α-tubulin [red], SPATA7-GFP, and DAPI). Bars: (main images) 10 µm; (insets) 2 µm. (b) Model displaying the CC zones PSTZ at the DCC and PCC that are visible in the Spata7 mutant retinae. The CC is comprised of PCC, a region homologous to the TZ of the primary cilium, and PSTZ, a specialized photoreceptor-specific zone of the CC that is crucial for maintenance of the CC microtubule core. OS, outer segment.
    Adult Spata7 Gfp Transgenic, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Roche)
    92
    Roche gfp
    Colocalization and effects of direct NPFFR2 signalling on <t>NPY</t> neurons. a Representative image of <t>GFP</t> expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p
    Gfp, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gfp centrin 2
    Primary cilium growth is asynchronous in sister cells, and independent of cytoplasmic differences (A) Sister pair of NIH/3T3 cells 6 h after mitotic shake-off in which the left cell has a cilium but the right cell does not; acetylated α-tubulin is red, <t>GFP-centrin</t> 2 is green, and DNA is blue. Bar, 10 μm. (B,C) Quantification of ciliogenesis in sister pairs in normal (B) or reduced-serum (0.5%) (C) media. N=300 cells for each timepoint; error bars, s.e.m. from 3 experiments. (D) Binucleate cell 8 h after mitotic shake-off, in which one centriole has a cilium; acetylated α-tubulin is red, GFP-centrin 2 is green, and DNA is blue. Bar, 10 μm. (E) Quantification of ciliogenesis in binucleate NIH/3T3 cells. n=300 cells for each timepoint; error bars, s.e.m. from 3 experiments.
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    SPATA7 extends AHI1 localization apical of the TZ in primary cilia. (a) Endogenous AHI1 (white) localizes at the TZ of the primary cilium, marked by acetylated α-tubulin in hTERT-RPE1 cells (left). Ectopic expression of hSPATA7-GFP (right) induces apical extension of the endogenous AHI1 localization signal beyond the TZ into the ciliary axoneme (subsets display zoomed images of AHI1 [white], acetylated α-tubulin [red], SPATA7-GFP, and DAPI). Bars: (main images) 10 µm; (insets) 2 µm. (b) Model displaying the CC zones PSTZ at the DCC and PCC that are visible in the Spata7 mutant retinae. The CC is comprised of PCC, a region homologous to the TZ of the primary cilium, and PSTZ, a specialized photoreceptor-specific zone of the CC that is crucial for maintenance of the CC microtubule core. OS, outer segment.

    Journal: The Journal of Cell Biology

    Article Title: SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium

    doi: 10.1083/jcb.201712117

    Figure Lengend Snippet: SPATA7 extends AHI1 localization apical of the TZ in primary cilia. (a) Endogenous AHI1 (white) localizes at the TZ of the primary cilium, marked by acetylated α-tubulin in hTERT-RPE1 cells (left). Ectopic expression of hSPATA7-GFP (right) induces apical extension of the endogenous AHI1 localization signal beyond the TZ into the ciliary axoneme (subsets display zoomed images of AHI1 [white], acetylated α-tubulin [red], SPATA7-GFP, and DAPI). Bars: (main images) 10 µm; (insets) 2 µm. (b) Model displaying the CC zones PSTZ at the DCC and PCC that are visible in the Spata7 mutant retinae. The CC is comprised of PCC, a region homologous to the TZ of the primary cilium, and PSTZ, a specialized photoreceptor-specific zone of the CC that is crucial for maintenance of the CC microtubule core. OS, outer segment.

    Article Snippet: IP and liquid chromatography (LC)–tandem MS (MS/MS) analysis For coimmunoprecipitation experiments, retinae from five adult Spata7 -GFP transgenic and five adult WT mice (per replicate across three replicates) were lysed in the IP buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, and protease inhibitor cocktail tablets [11836170007; Roche]) by homogenization followed by sonication.

    Techniques: Expressing, Droplet Countercurrent Chromatography, Periodic Counter-current Chromatography, Mutagenesis

    SPATA7 interacts with members of the NPHP–RPGR complex. (a) Distribution of mean GFP/WT Skyline peptide fold change of top candidate interacting partners of SPATA7 observed over three repeats. The asterisk signifies candidates that are enriched > 1,000-fold in the GFP IP fraction in comparison with the WT control fraction. (b) The interaction between SPATA7 and representative RPGR was confirmed in vitro by the BiFC assay. HEK293T cells were transfected with either SPATA7-CVN alone (top) or together with RPGR-VC constructs (bottom). Strong fluorescence signals (green) were observed in cells expressing both SPATA7 and RPGR, which are quantified in c. Bar, 20 µm. (c) The percentage of BiFC-positive cells for all candidate-interacting partners that are known TZ module members was quantified by FACS ( t test) in comparison with untransfected cells and SPATA7 alone. Error bars show means ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium

    doi: 10.1083/jcb.201712117

    Figure Lengend Snippet: SPATA7 interacts with members of the NPHP–RPGR complex. (a) Distribution of mean GFP/WT Skyline peptide fold change of top candidate interacting partners of SPATA7 observed over three repeats. The asterisk signifies candidates that are enriched > 1,000-fold in the GFP IP fraction in comparison with the WT control fraction. (b) The interaction between SPATA7 and representative RPGR was confirmed in vitro by the BiFC assay. HEK293T cells were transfected with either SPATA7-CVN alone (top) or together with RPGR-VC constructs (bottom). Strong fluorescence signals (green) were observed in cells expressing both SPATA7 and RPGR, which are quantified in c. Bar, 20 µm. (c) The percentage of BiFC-positive cells for all candidate-interacting partners that are known TZ module members was quantified by FACS ( t test) in comparison with untransfected cells and SPATA7 alone. Error bars show means ± SEM.

    Article Snippet: IP and liquid chromatography (LC)–tandem MS (MS/MS) analysis For coimmunoprecipitation experiments, retinae from five adult Spata7 -GFP transgenic and five adult WT mice (per replicate across three replicates) were lysed in the IP buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, and protease inhibitor cocktail tablets [11836170007; Roche]) by homogenization followed by sonication.

    Techniques: In Vitro, Bimolecular Fluorescence Complementation Assay, Transfection, Construct, Fluorescence, Expressing, FACS

    Colocalization and effects of direct NPFFR2 signalling on NPY neurons. a Representative image of GFP expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p

    Journal: Nature Communications

    Article Title: Diet-induced adaptive thermogenesis requires neuropeptide FF receptor-2 signalling

    doi: 10.1038/s41467-018-06462-0

    Figure Lengend Snippet: Colocalization and effects of direct NPFFR2 signalling on NPY neurons. a Representative image of GFP expression in the Arc of a NPY-TRAP mouse brain. Scale bar = 100 µm. b, c Quantification of the expression of Npy and Npffr2 mRNA in the input and immunoprecipitated (IP) RNA isolated from the Arc of NPY-TRAP ( n = 10), Ins-TRAP ( n = 3) and WT-TRAP ( n = 3) mice. One-way ANOVA was used to determine difference between groups. ∗∗ p

    Article Snippet: RT-qPCR using primers for Npy , GFP and Npffr2 was carried out in samples prior (input) and after the immunoprecipitation in at least triplicates from 1:5 dilution cDNA from each sample using the LightCycler® (LightCycler® 480 Real-Time PCR system, Roche Applied Science, Germany), SYBR Green I (Molecular Probes) and Platinum Taq DNA Polymerase (Invitrogen).

    Techniques: Expressing, Immunoprecipitation, Isolation, Mouse Assay

    The MAPK-binding site on MKP-1 contributes to nuclear accumulation. (A) Schematic representation of the basic cluster (RRR) on MKP-1 that resides between the CH2A and CH2B domains. This RRR basic cluster was mutated to ASA in the context of GFP-MKP-1

    Journal:

    Article Title: The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †

    doi: 10.1128/MCB.25.11.4792-4803.2005

    Figure Lengend Snippet: The MAPK-binding site on MKP-1 contributes to nuclear accumulation. (A) Schematic representation of the basic cluster (RRR) on MKP-1 that resides between the CH2A and CH2B domains. This RRR basic cluster was mutated to ASA in the context of GFP-MKP-1

    Article Snippet: Immortalized MKP-1 MEFs were cotransfected with 5XSRE-luciferase (0.25 to 0.5 μg) and pRL- Renilla (25 ng) along with 4 μg GFP, 1 μg GFP-MKP-1, and 3 μg GFP or 1 μg GFP-MKP-11-136 , and 3 μg GFP with FuGENE-6 (Roche) according to the manufacturer's instructions.

    Techniques: Binding Assay

    The NH 2 terminus of MKP-1 is required for nuclear localization. (A to D) COS-7 cells were transiently transfected with GFP-MKP-1 (A), GFP- MKP-1 47-367 (B), GFP-MKP-1 Δ47-136 (C), or GFP-MKP-1 137-367 (D). Confocal imaging was used to visualize GFP

    Journal:

    Article Title: The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †

    doi: 10.1128/MCB.25.11.4792-4803.2005

    Figure Lengend Snippet: The NH 2 terminus of MKP-1 is required for nuclear localization. (A to D) COS-7 cells were transiently transfected with GFP-MKP-1 (A), GFP- MKP-1 47-367 (B), GFP-MKP-1 Δ47-136 (C), or GFP-MKP-1 137-367 (D). Confocal imaging was used to visualize GFP

    Article Snippet: Immortalized MKP-1 MEFs were cotransfected with 5XSRE-luciferase (0.25 to 0.5 μg) and pRL- Renilla (25 ng) along with 4 μg GFP, 1 μg GFP-MKP-1, and 3 μg GFP or 1 μg GFP-MKP-11-136 , and 3 μg GFP with FuGENE-6 (Roche) according to the manufacturer's instructions.

    Techniques: Transfection, Imaging

    Generation of GFP-MKP-1 fusion proteins. (A) Schematic representation of the GFP fusion proteins of MKP-1 used in this study. (B) COS-7 cells were either left untransfected or were transfected with expression vectors encoding the GFP-MKP-1 fusion proteins

    Journal:

    Article Title: The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †

    doi: 10.1128/MCB.25.11.4792-4803.2005

    Figure Lengend Snippet: Generation of GFP-MKP-1 fusion proteins. (A) Schematic representation of the GFP fusion proteins of MKP-1 used in this study. (B) COS-7 cells were either left untransfected or were transfected with expression vectors encoding the GFP-MKP-1 fusion proteins

    Article Snippet: Immortalized MKP-1 MEFs were cotransfected with 5XSRE-luciferase (0.25 to 0.5 μg) and pRL- Renilla (25 ng) along with 4 μg GFP, 1 μg GFP-MKP-1, and 3 μg GFP or 1 μg GFP-MKP-11-136 , and 3 μg GFP with FuGENE-6 (Roche) according to the manufacturer's instructions.

    Techniques: Transfection, Expressing

    The NH 2 terminus of MKP-1 is sufficient for nuclear targeting. GFP-MKP-1 1-46 (A), GFP-MKP-1 47-136 (B), and GFP-MKP-1 1-136 (C) were transiently transfected into COS-7 cells, and confocal imaging was performed to visualize for GFP. The graphs below each

    Journal:

    Article Title: The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †

    doi: 10.1128/MCB.25.11.4792-4803.2005

    Figure Lengend Snippet: The NH 2 terminus of MKP-1 is sufficient for nuclear targeting. GFP-MKP-1 1-46 (A), GFP-MKP-1 47-136 (B), and GFP-MKP-1 1-136 (C) were transiently transfected into COS-7 cells, and confocal imaging was performed to visualize for GFP. The graphs below each

    Article Snippet: Immortalized MKP-1 MEFs were cotransfected with 5XSRE-luciferase (0.25 to 0.5 μg) and pRL- Renilla (25 ng) along with 4 μg GFP, 1 μg GFP-MKP-1, and 3 μg GFP or 1 μg GFP-MKP-11-136 , and 3 μg GFP with FuGENE-6 (Roche) according to the manufacturer's instructions.

    Techniques: Transfection, Imaging

    The NH 2 terminus of MKP-1 inhibits SRE-mediated activation by preventing Elk-1 phosphorylation. (A) Serum-deprived 293 cells expressing the indicated GFP-MKP-1 fusion proteins along with Flag-Elk-1 were stimulated with 10% FBS for 30 min. Whole-cell lysates

    Journal:

    Article Title: The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Nuclear Targeting and Serum Response Element Transcriptional Regulation †

    doi: 10.1128/MCB.25.11.4792-4803.2005

    Figure Lengend Snippet: The NH 2 terminus of MKP-1 inhibits SRE-mediated activation by preventing Elk-1 phosphorylation. (A) Serum-deprived 293 cells expressing the indicated GFP-MKP-1 fusion proteins along with Flag-Elk-1 were stimulated with 10% FBS for 30 min. Whole-cell lysates

    Article Snippet: Immortalized MKP-1 MEFs were cotransfected with 5XSRE-luciferase (0.25 to 0.5 μg) and pRL- Renilla (25 ng) along with 4 μg GFP, 1 μg GFP-MKP-1, and 3 μg GFP or 1 μg GFP-MKP-11-136 , and 3 μg GFP with FuGENE-6 (Roche) according to the manufacturer's instructions.

    Techniques: Activation Assay, Expressing

    Primary cilium growth is asynchronous in sister cells, and independent of cytoplasmic differences (A) Sister pair of NIH/3T3 cells 6 h after mitotic shake-off in which the left cell has a cilium but the right cell does not; acetylated α-tubulin is red, GFP-centrin 2 is green, and DNA is blue. Bar, 10 μm. (B,C) Quantification of ciliogenesis in sister pairs in normal (B) or reduced-serum (0.5%) (C) media. N=300 cells for each timepoint; error bars, s.e.m. from 3 experiments. (D) Binucleate cell 8 h after mitotic shake-off, in which one centriole has a cilium; acetylated α-tubulin is red, GFP-centrin 2 is green, and DNA is blue. Bar, 10 μm. (E) Quantification of ciliogenesis in binucleate NIH/3T3 cells. n=300 cells for each timepoint; error bars, s.e.m. from 3 experiments.

    Journal: Current Biology

    Article Title: Centriole Age Underlies Asynchronous Primary Cilium Growth in Mammalian Cells

    doi: 10.1016/j.cub.2009.07.034

    Figure Lengend Snippet: Primary cilium growth is asynchronous in sister cells, and independent of cytoplasmic differences (A) Sister pair of NIH/3T3 cells 6 h after mitotic shake-off in which the left cell has a cilium but the right cell does not; acetylated α-tubulin is red, GFP-centrin 2 is green, and DNA is blue. Bar, 10 μm. (B,C) Quantification of ciliogenesis in sister pairs in normal (B) or reduced-serum (0.5%) (C) media. N=300 cells for each timepoint; error bars, s.e.m. from 3 experiments. (D) Binucleate cell 8 h after mitotic shake-off, in which one centriole has a cilium; acetylated α-tubulin is red, GFP-centrin 2 is green, and DNA is blue. Bar, 10 μm. (E) Quantification of ciliogenesis in binucleate NIH/3T3 cells. n=300 cells for each timepoint; error bars, s.e.m. from 3 experiments.

    Article Snippet: NIH/3T3 lines stably expressing GFP-centrin 2 or tdTomato-inversin were generated by transfection of pTS1175 or pTS1641 using Fugene 6 transfection reagent (Roche) followed by selection with 450 μg/ml Geneticin (Invitrogen) and clonal isolation.

    Techniques:

    The timing of new mother centriole maturation correlates with asynchronous cilium growth (A) NIH/3T3 cells in mitosis and early G1 in which one mother centriole (left side of each panel) stains more brightly for cenexin/ODF2 than the other. Cenexin/ODF2 is shown alone in the top panels; in the bottom panels, acetylated α-tubulin is red, cenexin/ODF2 is green, and DNA is blue. Bar, 10 μm. In all panels, both centrosomes were in the same focal plane. (B,D) Cells after 18 h etoposide treatment in which both mother centrioles possess primary cilia; acetylated α-tubulin is red, GFP-centrin 2 is green in (B), cenexin/ODF2 is green in (D), and DNA is blue. Bars, 10 μm. (C) Quantitation of the fraction of cells with four centrioles that have 0, 1 or 2 cilia before and after the 18 h etoposide treatment.

    Journal: Current Biology

    Article Title: Centriole Age Underlies Asynchronous Primary Cilium Growth in Mammalian Cells

    doi: 10.1016/j.cub.2009.07.034

    Figure Lengend Snippet: The timing of new mother centriole maturation correlates with asynchronous cilium growth (A) NIH/3T3 cells in mitosis and early G1 in which one mother centriole (left side of each panel) stains more brightly for cenexin/ODF2 than the other. Cenexin/ODF2 is shown alone in the top panels; in the bottom panels, acetylated α-tubulin is red, cenexin/ODF2 is green, and DNA is blue. Bar, 10 μm. In all panels, both centrosomes were in the same focal plane. (B,D) Cells after 18 h etoposide treatment in which both mother centrioles possess primary cilia; acetylated α-tubulin is red, GFP-centrin 2 is green in (B), cenexin/ODF2 is green in (D), and DNA is blue. Bars, 10 μm. (C) Quantitation of the fraction of cells with four centrioles that have 0, 1 or 2 cilia before and after the 18 h etoposide treatment.

    Article Snippet: NIH/3T3 lines stably expressing GFP-centrin 2 or tdTomato-inversin were generated by transfection of pTS1175 or pTS1641 using Fugene 6 transfection reagent (Roche) followed by selection with 450 μg/ml Geneticin (Invitrogen) and clonal isolation.

    Techniques: Quantitation Assay