rabbit anti-gfp serum Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher rabbit anti gfp serum
    Recombinant CPs are related to the HLR in Samsun nn. (A) Structure diagrams of recombinant <t>TMV</t> CP and <t>GFP</t> proteins. Foreign peptides were fused in frame to the site S 154 -G 155 near the C-terminus of TMV CP to generate recombinant CP proteins. GFP was inserted in frame downstream of the N-terminal 18 amino acid residues to replace CP. Foreign peptides were then fused to the C-terminus of GFP to generate recombinant GFP proteins. CP, coat protein. GFP, Green fluorescent protein. FE, foreign epitope. Total proteins were extracted from tobacco seedlings at 4 dpi with wt TMV, TMVΔcpGFP, TMVΔcpGFPFN20, TMVΔcpGFPSC1754 (B), TMVFN20 and TMVSC1754 (C) at 25°C, respectively. Expressed recombinant proteins were separated on 12% SDS-PAGE and visualized by using anti-GFP antibody or anti-CP antibody in western analysis. A protein standard marker (Precision Plus Protein™ standards, Bio-Rad, U.S.A.) was used to measure the sizes of recombinant proteins. Phenotypes of inoculated leaves of Samsun nn were photographed at the same time. Infection buffer was used as negative control.
    Rabbit Anti Gfp Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti gfp serum/product/Thermo Fisher
    Average 99 stars, based on 171 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gfp serum - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore rabbit anti gfp serum
    Activation of UbDha in vivo a) In vivo labeling of endogenous E1 enzymes with Cy5-UbDha. Fluorescence scanning and <t>immunoblotting</t> of lysates from HeLa cell electroporated with the probe and harvested at indicated time intervals following electroporation with the probe. b) In vivo labeling of UBE1 with Cy5-UbDha following UBE1 inhibitor PYR-41 (50 µM) treatment. Fluorescence scan and quantification (% labeling in the absence of PYR-41; n=3, error bars correspond to SD, with significance (p) assessed using a two-sided t-test) are shown. c ) Distribution of Cy5-UbDha (magenta) in cells ectopically expressing <t>GFP-UBE1</t> (green) relative to untransfected cells. Representative 3D confocal compilations of fixed cells treated as indicated are shown with DAPI (blue) overlays and nuclear insets; scale bars = 10 µm. d) Pixel traces of DAPI and Cy5-UbDha (marked with dotted lines in d) plotted as fluorescence over distance. e) .
    Rabbit Anti Gfp Serum, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti gfp serum/product/Millipore
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gfp serum - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    98
    Abcam rabbit anti gfp serum
    Immunogold Localization of <t>VHA-a1–GFP</t> and TGN Morphology.
    Rabbit Anti Gfp Serum, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti gfp serum/product/Abcam
    Average 98 stars, based on 73 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gfp serum - by Bioz Stars, 2020-09
    98/100 stars
      Buy from Supplier

    99
    Thermo Fisher gfp tag polyclonal antibody
    Cleavage of wild-type (WT) and mutant HIV-1 <t>Tat-GFP</t> proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 <t>polyclonal</t> antibody as indicated.
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8760 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp tag polyclonal antibody/product/Thermo Fisher
    Average 99 stars, based on 8760 article reviews
    Price from $9.99 to $1999.99
    gfp tag polyclonal antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Abcam anti gfp antibody chip grade
    Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of <t>P[acman]-EGFP-dsh</t> . Wings immunolabelled for <t>GFP</t> (green), or Stbm (red), and clones marked by loss of β-gal (blue).
    Anti Gfp Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gfp antibody chip grade/product/Abcam
    Average 99 stars, based on 505 article reviews
    Price from $9.99 to $1999.99
    anti gfp antibody chip grade - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Rockland Immunochemicals biotin conjugated goat polyclonal anti gfp
    Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of <t>P[acman]-EGFP-dsh</t> . Wings immunolabelled for <t>GFP</t> (green), or Stbm (red), and clones marked by loss of β-gal (blue).
    Biotin Conjugated Goat Polyclonal Anti Gfp, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotin conjugated goat polyclonal anti gfp/product/Rockland Immunochemicals
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    biotin conjugated goat polyclonal anti gfp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    97
    Rockland Immunochemicals goat anti gfp igg
    Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of <t>P[acman]-EGFP-dsh</t> . Wings immunolabelled for <t>GFP</t> (green), or Stbm (red), and clones marked by loss of β-gal (blue).
    Goat Anti Gfp Igg, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 97/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti gfp igg/product/Rockland Immunochemicals
    Average 97 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    goat anti gfp igg - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    90
    Exbio Praha rabbit anti gfp serum
    Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of <t>P[acman]-EGFP-dsh</t> . Wings immunolabelled for <t>GFP</t> (green), or Stbm (red), and clones marked by loss of β-gal (blue).
    Rabbit Anti Gfp Serum, supplied by Exbio Praha, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti gfp serum/product/Exbio Praha
    Average 90 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gfp serum - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    85
    Thermo Fisher polyclonal anti gfp rabbit serum
    Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of <t>P[acman]-EGFP-dsh</t> . Wings immunolabelled for <t>GFP</t> (green), or Stbm (red), and clones marked by loss of β-gal (blue).
    Polyclonal Anti Gfp Rabbit Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti gfp rabbit serum/product/Thermo Fisher
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti gfp rabbit serum - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    99
    Thermo Fisher anti gfp antibodies
    ParB spreads from parS (−1.6°) into comCDE . In the strains of interest, ParB was replaced by a <t>ParB-GFP</t> fusion. Exponentially growing cells were subjected to chromatin <t>immunoprecipitation</t> (ChIP) using anti-GFP antibodies, and the pulled-down DNA was subsequently analyzed by qPCR. (A and B) The WT (DLA42) versus the parS (−1.6°) mut strain (DLA43). (C and D) The parS (−1.6°) mut C strain (DLA77) versus the parS (−1.6°) mut NC strain (DLA80). The loci amplified by primer pairs 1, 2, 3, and 4 are shown in blue in Fig. 3A . The primer pair P amplifies another parS site [ parS (+2°)] situated +11 kb from oriC . Primer pairs T1 and T2 amplify 2 different loci in the terminus region. The graphs show pulldown efficiency (ChIP-DNA/input DNA × 100) for each primer pair. Note the different y axis scale between panels A and C and panels B and D.
    Anti Gfp Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gfp antibodies/product/Thermo Fisher
    Average 99 stars, based on 719 article reviews
    Price from $9.99 to $1999.99
    anti gfp antibodies - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Recombinant CPs are related to the HLR in Samsun nn. (A) Structure diagrams of recombinant TMV CP and GFP proteins. Foreign peptides were fused in frame to the site S 154 -G 155 near the C-terminus of TMV CP to generate recombinant CP proteins. GFP was inserted in frame downstream of the N-terminal 18 amino acid residues to replace CP. Foreign peptides were then fused to the C-terminus of GFP to generate recombinant GFP proteins. CP, coat protein. GFP, Green fluorescent protein. FE, foreign epitope. Total proteins were extracted from tobacco seedlings at 4 dpi with wt TMV, TMVΔcpGFP, TMVΔcpGFPFN20, TMVΔcpGFPSC1754 (B), TMVFN20 and TMVSC1754 (C) at 25°C, respectively. Expressed recombinant proteins were separated on 12% SDS-PAGE and visualized by using anti-GFP antibody or anti-CP antibody in western analysis. A protein standard marker (Precision Plus Protein™ standards, Bio-Rad, U.S.A.) was used to measure the sizes of recombinant proteins. Phenotypes of inoculated leaves of Samsun nn were photographed at the same time. Infection buffer was used as negative control.

    Journal: PLoS ONE

    Article Title: An Induced Hypersensitive-Like Response Limits Expression of Foreign Peptides via a Recombinant TMV-Based Vector in a Susceptible Tobacco

    doi: 10.1371/journal.pone.0015087

    Figure Lengend Snippet: Recombinant CPs are related to the HLR in Samsun nn. (A) Structure diagrams of recombinant TMV CP and GFP proteins. Foreign peptides were fused in frame to the site S 154 -G 155 near the C-terminus of TMV CP to generate recombinant CP proteins. GFP was inserted in frame downstream of the N-terminal 18 amino acid residues to replace CP. Foreign peptides were then fused to the C-terminus of GFP to generate recombinant GFP proteins. CP, coat protein. GFP, Green fluorescent protein. FE, foreign epitope. Total proteins were extracted from tobacco seedlings at 4 dpi with wt TMV, TMVΔcpGFP, TMVΔcpGFPFN20, TMVΔcpGFPSC1754 (B), TMVFN20 and TMVSC1754 (C) at 25°C, respectively. Expressed recombinant proteins were separated on 12% SDS-PAGE and visualized by using anti-GFP antibody or anti-CP antibody in western analysis. A protein standard marker (Precision Plus Protein™ standards, Bio-Rad, U.S.A.) was used to measure the sizes of recombinant proteins. Phenotypes of inoculated leaves of Samsun nn were photographed at the same time. Infection buffer was used as negative control.

    Article Snippet: After being incubated in a blocking buffer (5% skim milk, 20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h, the PVDF membrane was incubated with rabbit anti-TMV CP serum (1∶500 dilution in the blocking buffer) or with rabbit anti-GFP serum (1∶1000 dilution, Invitrogen, U.S.A.) for 1 h, followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (1∶10,000 dilution, Pierce, U.S.A.) for 1 h, and then developed with a chemiluminescence system (ECL, Amersham, U.S.A.).

    Techniques: Recombinant, SDS Page, Western Blot, Marker, Infection, Negative Control

    Stability of SpoVE is dependent on SpoVD A. SpoVE-GFP is unstable in a spoVD mutant. Strains JDB1933 (wt) and JDB2148 ( spoVD ) were sporulated by resuspension. Samples taken at each time point were normalized to an OD 600 and lysates were subjected to 13.5% SDS-PAGE and probed with anti-GFP followed by anti-rabbit-HRP and detected by ECL. The arrowhead indicates full-length product, while the asterisk indicates the main cleavage product. B. SEDS proteins require their specific PBP for stability when expressed in a heterologous system. E. coli BL21 strains were induced for 2h with 0.1% arabinose at 25°C. Pellets containing equivalent amounts of cells (as by determined OD 600 ) were solubilized in 1x sample buffer at RT and subjected to 13.5% SDS-PAGE. After transfer to nitrocellulose, the blot was probed with anti-GFP, anti-rabbit-HRP, ECL plus, then stripped and probed with anti-GST, anti-rabbit-HRP to verify GST fusion expression. Full-length product is indicated by an arrowhead and the main cleavage product is indicated by an asterisk. SpoVE-GFP is stabilized by GST-SpoVD expression, but not by GST-PBP2B. FtsW-GFP is stabilized by GST-PBP2B, but not by GST-SpoVD.

    Journal: Journal of molecular biology

    Article Title: Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

    doi: 10.1016/j.jmb.2010.04.036

    Figure Lengend Snippet: Stability of SpoVE is dependent on SpoVD A. SpoVE-GFP is unstable in a spoVD mutant. Strains JDB1933 (wt) and JDB2148 ( spoVD ) were sporulated by resuspension. Samples taken at each time point were normalized to an OD 600 and lysates were subjected to 13.5% SDS-PAGE and probed with anti-GFP followed by anti-rabbit-HRP and detected by ECL. The arrowhead indicates full-length product, while the asterisk indicates the main cleavage product. B. SEDS proteins require their specific PBP for stability when expressed in a heterologous system. E. coli BL21 strains were induced for 2h with 0.1% arabinose at 25°C. Pellets containing equivalent amounts of cells (as by determined OD 600 ) were solubilized in 1x sample buffer at RT and subjected to 13.5% SDS-PAGE. After transfer to nitrocellulose, the blot was probed with anti-GFP, anti-rabbit-HRP, ECL plus, then stripped and probed with anti-GST, anti-rabbit-HRP to verify GST fusion expression. Full-length product is indicated by an arrowhead and the main cleavage product is indicated by an asterisk. SpoVE-GFP is stabilized by GST-SpoVD expression, but not by GST-PBP2B. FtsW-GFP is stabilized by GST-PBP2B, but not by GST-SpoVD.

    Article Snippet: Gels were transferred to Biotrace-NT (Pall) and probed using anti-GFP rabbit serum (gift of H. Shuman, 1:25000), anti-rabbit-HRP (1:25000, Pierce), then ECL plus (GE).

    Techniques: Mutagenesis, SDS Page, Expressing

    Interaction between SpoVE and SpoVD A. SpoVE-GFP co-immunoprecipitates with SpoVD-FLAG in B. subtilis . Strains expressing combinations of GFP and FLAG fusion proteins were sporulated by resuspension and lysates from samples taken at T2.5 were solubilized in 1% NP-40 and immunoprecipitated with M2 Affinity resin (Sigma). Lysates and eluates were subjected to 13.5% SDS-PAGE, and blots were probed with anti-GFP (top and bottom) or anti-FLAG (middle), anti-rabbit-HRP (Pierce), and detected with ECL. SpoVD-FLAG co-immunoprecipitated SpoVEGFP (lane 3; JDB1676) but not MalF-GFP (lane 2; JDB1665) or FtsW-GFP (lane 4; JDB1885). SecY-FLAG did not co-immunoprecipitate SpoVE-GFP (lane 1; JDB2155). B. SpoVE-GFP co-affinity purifies with GST-SpoVD. E. coli BL21 strains expressing GFP and GST tagged proteins were grown in LB at 37°C and induced for 120 min with 0.1% arabinose at OD 600 =0.8. Samples were collected and lysates containing equivalent amounts of cells (as by determined OD 600 ) were solubilized with 1% NP-40 before affinity purification using immobilized glutathione. Lysates and eluates were subjected to 13.5% SDS-PAGE. After transfer to nitrocellulose, the blots were probed with anti-GFP and anti-rabbit-HRP (top and bottom), then stripped and probed with anti-GST and anti-rabbit-HRP (middle). GST-SpoVD co-affinity purified with SpoVE-GFP (lane 3; JDE1043) but not GFP alone (lane 1; JDE1045). In addition, GST alone did not co-affinity purify SpoVE-GFP (lane 2; JDE1044).

    Journal: Journal of molecular biology

    Article Title: Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

    doi: 10.1016/j.jmb.2010.04.036

    Figure Lengend Snippet: Interaction between SpoVE and SpoVD A. SpoVE-GFP co-immunoprecipitates with SpoVD-FLAG in B. subtilis . Strains expressing combinations of GFP and FLAG fusion proteins were sporulated by resuspension and lysates from samples taken at T2.5 were solubilized in 1% NP-40 and immunoprecipitated with M2 Affinity resin (Sigma). Lysates and eluates were subjected to 13.5% SDS-PAGE, and blots were probed with anti-GFP (top and bottom) or anti-FLAG (middle), anti-rabbit-HRP (Pierce), and detected with ECL. SpoVD-FLAG co-immunoprecipitated SpoVEGFP (lane 3; JDB1676) but not MalF-GFP (lane 2; JDB1665) or FtsW-GFP (lane 4; JDB1885). SecY-FLAG did not co-immunoprecipitate SpoVE-GFP (lane 1; JDB2155). B. SpoVE-GFP co-affinity purifies with GST-SpoVD. E. coli BL21 strains expressing GFP and GST tagged proteins were grown in LB at 37°C and induced for 120 min with 0.1% arabinose at OD 600 =0.8. Samples were collected and lysates containing equivalent amounts of cells (as by determined OD 600 ) were solubilized with 1% NP-40 before affinity purification using immobilized glutathione. Lysates and eluates were subjected to 13.5% SDS-PAGE. After transfer to nitrocellulose, the blots were probed with anti-GFP and anti-rabbit-HRP (top and bottom), then stripped and probed with anti-GST and anti-rabbit-HRP (middle). GST-SpoVD co-affinity purified with SpoVE-GFP (lane 3; JDE1043) but not GFP alone (lane 1; JDE1045). In addition, GST alone did not co-affinity purify SpoVE-GFP (lane 2; JDE1044).

    Article Snippet: Gels were transferred to Biotrace-NT (Pall) and probed using anti-GFP rabbit serum (gift of H. Shuman, 1:25000), anti-rabbit-HRP (1:25000, Pierce), then ECL plus (GE).

    Techniques: Expressing, Immunoprecipitation, SDS Page, Affinity Purification

    Expression and stability of MuSK mutants in HEK 293 cells. ( A ) A western blot analysis in cells co-transfected with WT or MUSK mutants and GFP using a polyclonal anti-MuSK goat antibody demonstrates reduced expression of MuSK in cells transfected with M605I and A727V in comparison with cells transfected with the WT construct (82% of WT for M605I and 87% of WT for A727V ). The expression of GFP, tested with an anti-GFP polyclonal rabbit antibody, was similar in cells transfected with WT and MUSK mutants. ( B ) Analysis conducted in cells exposed to cycloheximide at set times intervals: 0, 30, 60 and 90 min, 36 h after transfection with WT or MUSK mutants revealed decreased expression of MuSK in cells transfected with the mutants compared with cells transfected with the WT construct. At time 0, expression of M605 I was 87%, whereas A727V was 63% compared with WT. However, there were no major differences in the rate of degradation of MuSK in cells transfected with WT or MUSK mutant constructs. Expression of GAPDH detected with an anti-GAPDH monoclonal antibody showed no reduction of GAPDH expression in all the samples.

    Journal: Human Molecular Genetics

    Article Title: Mutations in MUSK causing congenital myasthenic syndrome impair MuSK-Dok-7 interaction

    doi: 10.1093/hmg/ddq110

    Figure Lengend Snippet: Expression and stability of MuSK mutants in HEK 293 cells. ( A ) A western blot analysis in cells co-transfected with WT or MUSK mutants and GFP using a polyclonal anti-MuSK goat antibody demonstrates reduced expression of MuSK in cells transfected with M605I and A727V in comparison with cells transfected with the WT construct (82% of WT for M605I and 87% of WT for A727V ). The expression of GFP, tested with an anti-GFP polyclonal rabbit antibody, was similar in cells transfected with WT and MUSK mutants. ( B ) Analysis conducted in cells exposed to cycloheximide at set times intervals: 0, 30, 60 and 90 min, 36 h after transfection with WT or MUSK mutants revealed decreased expression of MuSK in cells transfected with the mutants compared with cells transfected with the WT construct. At time 0, expression of M605 I was 87%, whereas A727V was 63% compared with WT. However, there were no major differences in the rate of degradation of MuSK in cells transfected with WT or MUSK mutant constructs. Expression of GAPDH detected with an anti-GAPDH monoclonal antibody showed no reduction of GAPDH expression in all the samples.

    Article Snippet: For the expression studies, the blot was also probed with a primary anti-GFP rabbit polyclonal serum (1:1000; Invitrogen) followed by a polyclonal goat anti-rabbit IgG-HRP secondary antibody (1:2000; Dako North America, Inc., Carpinteria, CA, USA) as a loading control.

    Techniques: Expressing, Western Blot, Transfection, Construct, Mutagenesis

    CPT1C enhances surface expression of GluA1. (A,B) Representative single confocal images of tsA201 cells co-expressing GluA1 and GFP (A) or GluA1 and CPT1C-GFP (B) . Surface GluA1 was labeled in live cells with anti-GluA1-NT and Alexafluor 555 (red signal in the images). Subsequently cells were permeabilized and total GluA1 expression level was labeled with the same primary antibody but with Alexafluor 647 (blue signal in the images). Scale bars: 20 μm. (C) Quantification of the GluA1 surface to total ratio normalized to GluA1, expressed as a percentage. GluA1 surface expression was increased by co-expression with CPT1C ( *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: CPT1C enhances surface expression of GluA1. (A,B) Representative single confocal images of tsA201 cells co-expressing GluA1 and GFP (A) or GluA1 and CPT1C-GFP (B) . Surface GluA1 was labeled in live cells with anti-GluA1-NT and Alexafluor 555 (red signal in the images). Subsequently cells were permeabilized and total GluA1 expression level was labeled with the same primary antibody but with Alexafluor 647 (blue signal in the images). Scale bars: 20 μm. (C) Quantification of the GluA1 surface to total ratio normalized to GluA1, expressed as a percentage. GluA1 surface expression was increased by co-expression with CPT1C ( *** p

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Expressing, Labeling

    GluA1 palmitoylation state is not altered by CPT1C and does not affect the interaction with GluA1. (A) Palmitoylation levels of GluA1 alone (GluA1) and together with CPT1C-GFP (GluA1+CPT1C), in transfected tsA201 cells, detected by means of Acyl-Biotin Exchange (ABE). The thiol-biotinylated immunoprecipitates of GluA1 following the ABE assay for both transfected conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 can only be detected in plus-hydroxilamine (+HAM) samples. Minus -HAM samples control non-specific incorporation of biotin. GluA1 palmitoylation levels (top) were detected by Western blotting with streptavidin-HRP (palmitoylation). After stripping the membranes the total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom). (B) Quantification of palmitoylation levels for GluA1 alone (open circles) or GluA1 plus CPT1C (filled circles) in tsA201 cells. Ratio of palmitoylated GluA1 to total GluA1 for each single experiment is shown together with mean (discontinuous horizontal lines) and SEM (continuous vertical lines) ( p > 0.05; Mann–Whitney U -test; n = 8 for both). (C) Co-IP of the membranous fraction of tsA201 cells co-expressing GluA1 wild type or non-palmitoylable mutants—GluA1(C585S), GluA1(C811S), and GluA1(C585,811S)—together with CPT1C-GFP. The interaction between CPT1C and GluA1 is not dependent on palmitoylation of C585 or C811 residues. As negative controls GluA1 was co-expressed with an empty plasmid expressing GFP alone (first lanes of the boxes) and CPT1C-GFP was co-expressed with an empty pDsRed (second lanes from the boxes). Transfected cells were lysed and membranes were solubilized as described in Figure 1 and methods. An input sample collected prior to immunoprecipitation of these extracts is shown as “INPUT.” Inputs and immunoprecipitated samples were separated and Western Blotted as described in Figure 1 . Immunoprecipitations were replicated three times.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: GluA1 palmitoylation state is not altered by CPT1C and does not affect the interaction with GluA1. (A) Palmitoylation levels of GluA1 alone (GluA1) and together with CPT1C-GFP (GluA1+CPT1C), in transfected tsA201 cells, detected by means of Acyl-Biotin Exchange (ABE). The thiol-biotinylated immunoprecipitates of GluA1 following the ABE assay for both transfected conditions were subjected to SDS-PAGE. Palmitoylation of GluA1 can only be detected in plus-hydroxilamine (+HAM) samples. Minus -HAM samples control non-specific incorporation of biotin. GluA1 palmitoylation levels (top) were detected by Western blotting with streptavidin-HRP (palmitoylation). After stripping the membranes the total amount of immunoprecipitated GluA1 was detected by Western blotting with anti-GluA1-NT antibody (anti-GluA1, bottom). (B) Quantification of palmitoylation levels for GluA1 alone (open circles) or GluA1 plus CPT1C (filled circles) in tsA201 cells. Ratio of palmitoylated GluA1 to total GluA1 for each single experiment is shown together with mean (discontinuous horizontal lines) and SEM (continuous vertical lines) ( p > 0.05; Mann–Whitney U -test; n = 8 for both). (C) Co-IP of the membranous fraction of tsA201 cells co-expressing GluA1 wild type or non-palmitoylable mutants—GluA1(C585S), GluA1(C811S), and GluA1(C585,811S)—together with CPT1C-GFP. The interaction between CPT1C and GluA1 is not dependent on palmitoylation of C585 or C811 residues. As negative controls GluA1 was co-expressed with an empty plasmid expressing GFP alone (first lanes of the boxes) and CPT1C-GFP was co-expressed with an empty pDsRed (second lanes from the boxes). Transfected cells were lysed and membranes were solubilized as described in Figure 1 and methods. An input sample collected prior to immunoprecipitation of these extracts is shown as “INPUT.” Inputs and immunoprecipitated samples were separated and Western Blotted as described in Figure 1 . Immunoprecipitations were replicated three times.

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Transfection, SDS Page, Western Blot, Stripping Membranes, Immunoprecipitation, MANN-WHITNEY, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation

    CPT1C specifically increases glutamate-evoked currents of GluA1-containing AMPARs. (A) Whole-cell current-voltage (IV) relationship for a tsA201 cell expressing GluA1 homomeric receptors. The IV plot was obtained by ramping membrane potential from −80 to +80 mV at a rate of 160 mV/ s in the presence of 1 mM glutamate plus 25 μM CTZ to avoid receptor desensitization. 100 μM spermine was added to the pipette solution. Inset represents the voltage protocol used. (B) Same as (A) but for a cell expressing GluA1 plus CPT1C-GFP (CPT1C). (C) Average normalized currents at -80 mV for GluA1 alone or together with CPT1C or CPT1A. GluA1 current density (-pA/pF) was increased by co-expression with CPT1C ( * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: CPT1C specifically increases glutamate-evoked currents of GluA1-containing AMPARs. (A) Whole-cell current-voltage (IV) relationship for a tsA201 cell expressing GluA1 homomeric receptors. The IV plot was obtained by ramping membrane potential from −80 to +80 mV at a rate of 160 mV/ s in the presence of 1 mM glutamate plus 25 μM CTZ to avoid receptor desensitization. 100 μM spermine was added to the pipette solution. Inset represents the voltage protocol used. (B) Same as (A) but for a cell expressing GluA1 plus CPT1C-GFP (CPT1C). (C) Average normalized currents at -80 mV for GluA1 alone or together with CPT1C or CPT1A. GluA1 current density (-pA/pF) was increased by co-expression with CPT1C ( * p

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Expressing, Transferring

    CPT1C does not alter AMPAR gating properties. (A) Current activated by rapid application of 10 mM glutamate (100 ms, −60 mV) to an outside-out patch from a tsA201 cell expressing GluA1 alone. Gray trace represents a single response and black line the average of 37 responses. Inset shows the current variance vs. mean current plot for this patch. The weighted mean single-channel conductance estimate for this record was 17.3 pS. (B) Same as (A) but for a cell expressing GluA1 plus CPT1C-GFP (CPT1C). Black line is the average of 38 responses. Inset: current variance vs. mean current plot for this patch giving a weighted mean single-channel conductance of 16.7 pS. (C–F) CPT1C has no influence on either weighted single channel conductance ( C ; pS), peak open probability ( D ; P o,peak ), desensitization kinetics ( E ; τ des ) or rectification index ( F ; I +60mV /I −60mV ) of GluA1 homomeric AMPARs. P > 0.05, Mann–Whitney U -test for all groups. Numbers in bars denote the number of recordings.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: CPT1C does not alter AMPAR gating properties. (A) Current activated by rapid application of 10 mM glutamate (100 ms, −60 mV) to an outside-out patch from a tsA201 cell expressing GluA1 alone. Gray trace represents a single response and black line the average of 37 responses. Inset shows the current variance vs. mean current plot for this patch. The weighted mean single-channel conductance estimate for this record was 17.3 pS. (B) Same as (A) but for a cell expressing GluA1 plus CPT1C-GFP (CPT1C). Black line is the average of 38 responses. Inset: current variance vs. mean current plot for this patch giving a weighted mean single-channel conductance of 16.7 pS. (C–F) CPT1C has no influence on either weighted single channel conductance ( C ; pS), peak open probability ( D ; P o,peak ), desensitization kinetics ( E ; τ des ) or rectification index ( F ; I +60mV /I −60mV ) of GluA1 homomeric AMPARs. P > 0.05, Mann–Whitney U -test for all groups. Numbers in bars denote the number of recordings.

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Mass Spectrometry, Expressing, MANN-WHITNEY

    GluA1 and GluA2 co-localize with CPT1C at intracellular compartments but not at the plasma membrane . CPT1C does not co-localize with the Golgi Apparatus marker GM-130. (A) Confocal image showing co-localization of CPT1C-GFP (green) with GluA1 (dark blue signal in left panel) in transfected tsA201 cells. Co-localization signal is displayed as light blue (left panel). There is no co-localization between CPT1C (green) and cell-surface GluA1 (red signal in right panel). Scale bar: 10 μm. (B) Confocal image showing co-localization of CPT1C (green) with GluA2 (dark blue signal in left panel) in transfected tsA201 cells. Co-localization signal is apparent as light blue color (left panel). There is no co-localization between CPT1C (green) and cell-surface GluA2 (red signal in right panel). Scale bar: 10 μm. (C) Co-localization (yellow signal in left panel) of CPT1C-GFP with ER marker (pDsRed-KDEL). Lack of co-localization (absence of yellow signal in right panel) of CPT1C (green) with the Golgi Apparatus marker GM-130 (red) in transfected tsA201 cells. Scale bars: 10 μm. (D) Representation of co-localization values quantified by Manders Overlap Coefficient (MOC; see Methods) expressed as mean ± SEM. MOC values for total GluAs and CPT1C (blue columns) were statistically different from surface GluAs and CPT1C (red columns; *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: GluA1 and GluA2 co-localize with CPT1C at intracellular compartments but not at the plasma membrane . CPT1C does not co-localize with the Golgi Apparatus marker GM-130. (A) Confocal image showing co-localization of CPT1C-GFP (green) with GluA1 (dark blue signal in left panel) in transfected tsA201 cells. Co-localization signal is displayed as light blue (left panel). There is no co-localization between CPT1C (green) and cell-surface GluA1 (red signal in right panel). Scale bar: 10 μm. (B) Confocal image showing co-localization of CPT1C (green) with GluA2 (dark blue signal in left panel) in transfected tsA201 cells. Co-localization signal is apparent as light blue color (left panel). There is no co-localization between CPT1C (green) and cell-surface GluA2 (red signal in right panel). Scale bar: 10 μm. (C) Co-localization (yellow signal in left panel) of CPT1C-GFP with ER marker (pDsRed-KDEL). Lack of co-localization (absence of yellow signal in right panel) of CPT1C (green) with the Golgi Apparatus marker GM-130 (red) in transfected tsA201 cells. Scale bars: 10 μm. (D) Representation of co-localization values quantified by Manders Overlap Coefficient (MOC; see Methods) expressed as mean ± SEM. MOC values for total GluAs and CPT1C (blue columns) were statistically different from surface GluAs and CPT1C (red columns; *** p

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Marker, Transfection

    GluA1 C585 is critical for the enhancement of current density and surface expression by CPT1C. (A–C) . Representative single confocal images of tsA201 cells co-expressing different versions of GluA1 with (right panels) or without (left panels) CPT1C-GFP. In the images, surface GluA1 is shown in red and total GluA1 in blue. Scale bar: 50 μm. (A) Native GluA1 co-expressing GFP (+GFP) as the control condition, or CPT1C-GFP (+CPT1C). (B) . Same as (A) but for cells expressing GluA1 containing the point mutation C585S that abolishes palmitoylation at this residue. (C) Same as (A,B) but for GluA1 with the point mutation C811S. (D) Quantification of the GluA1 surface to total ratio normalized to GluA1, expressed as a percentage. GluA1 surface expression was increased by co-expression of CPT1C for both GluA1 and GluA1(C811S) ( *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: GluA1 C585 is critical for the enhancement of current density and surface expression by CPT1C. (A–C) . Representative single confocal images of tsA201 cells co-expressing different versions of GluA1 with (right panels) or without (left panels) CPT1C-GFP. In the images, surface GluA1 is shown in red and total GluA1 in blue. Scale bar: 50 μm. (A) Native GluA1 co-expressing GFP (+GFP) as the control condition, or CPT1C-GFP (+CPT1C). (B) . Same as (A) but for cells expressing GluA1 containing the point mutation C585S that abolishes palmitoylation at this residue. (C) Same as (A,B) but for GluA1 with the point mutation C811S. (D) Quantification of the GluA1 surface to total ratio normalized to GluA1, expressed as a percentage. GluA1 surface expression was increased by co-expression of CPT1C for both GluA1 and GluA1(C811S) ( *** p

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Expressing, Mutagenesis

    GluA1 and GluA2 coimmunoprecipitate with CPT1C in expression systems. (A) Co-IP of the membranous fraction of tsA201 cells co-expressing either GluA1 alone or together with CPT1C-GFP confirming the interaction between CPT1C and GluA1. As a negative control GluA1 was co-expressed with an empty plasmid expressing GFP alone (pEGFP) and CPT1C-GFP was co-expressed with an empty plasmid (pcDNA3.0). Transfected cells were lysed and membranes were solubilized. 200-400 μg of solubilized membranes was immunoprecipitated with an anti-GFP antibody (IP: antiGFP) or with anti-GluA1-NT antibody (IP: antiGluA1). An input sample collected prior to immunoprecipitation of these extracts is shown as “INPUT.” Inputs and immunoprecipitated samples were separated using SDS-PAGE and Western Blot was performed using anti-GluA1-NT (WB: antiGluA1) or anti-GFP (WB: antiGFP) antibodies. Immunoprecipitations were performed three times. (B) Same as in (A) but for tsA201 cells expressing GluA2 or GluA2 plus CPT1C-GFP. Membrane lysates were immunoprecipitated with an anti-GFP antibody (IP: antiGFP) or a rabbit polyclonal anti-GluA2 (cytoplasmic domain) (IP: antiGluA2). Western Blots were performed using mouse anti-GluA2 (WB: antiGluA2) or anti-GFP (WB: antiGFP) antibodies. Immunoprecipitations were replicated three times.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

    doi: 10.3389/fncel.2014.00469

    Figure Lengend Snippet: GluA1 and GluA2 coimmunoprecipitate with CPT1C in expression systems. (A) Co-IP of the membranous fraction of tsA201 cells co-expressing either GluA1 alone or together with CPT1C-GFP confirming the interaction between CPT1C and GluA1. As a negative control GluA1 was co-expressed with an empty plasmid expressing GFP alone (pEGFP) and CPT1C-GFP was co-expressed with an empty plasmid (pcDNA3.0). Transfected cells were lysed and membranes were solubilized. 200-400 μg of solubilized membranes was immunoprecipitated with an anti-GFP antibody (IP: antiGFP) or with anti-GluA1-NT antibody (IP: antiGluA1). An input sample collected prior to immunoprecipitation of these extracts is shown as “INPUT.” Inputs and immunoprecipitated samples were separated using SDS-PAGE and Western Blot was performed using anti-GluA1-NT (WB: antiGluA1) or anti-GFP (WB: antiGFP) antibodies. Immunoprecipitations were performed three times. (B) Same as in (A) but for tsA201 cells expressing GluA2 or GluA2 plus CPT1C-GFP. Membrane lysates were immunoprecipitated with an anti-GFP antibody (IP: antiGFP) or a rabbit polyclonal anti-GluA2 (cytoplasmic domain) (IP: antiGluA2). Western Blots were performed using mouse anti-GluA2 (WB: antiGluA2) or anti-GFP (WB: antiGFP) antibodies. Immunoprecipitations were replicated three times.

    Article Snippet: 200–400 μg of total protein were incubated with 4 μg of antibody overnight at 4°C with orbital agitation (antibodies: mouse anti-GluA1-NT (N-terminus), rabbit anti-GluA2 (cytoplasmic domain) both from Merck Millipore, rabbit serum anti-GFP from Invitrogen).

    Techniques: Expressing, Co-Immunoprecipitation Assay, Negative Control, Plasmid Preparation, Transfection, Immunoprecipitation, SDS Page, Western Blot

    STAC3 colocalizes also with heterologous Ca V 1.2 in skeletal muscle triads. ( A ) When co-expressed with Ca V 1.2, STAC3 is colocalized in clusters with the RyR and the Ca V subunits Ca V 1.2 and β 1a in dysgenic myotubes. Color overlay: 4X magnification of blue rectangle. Scale bars: 10 μm and 5 μm. ( B ) FRAP analysis of STAC3-GFP co-expressed with Ca V 1.2 shows very little recovery of fluorescence, very similar to when STAC3 is co-expressed with Ca V 1.1. Average recovery curves reveal similar low fluorescence recovery of STAC3-GFP co-expressed with Ca V 1.2 (red) and with Ca V 1.1 (blue) and comparable recovery rates 75 s after bleaching (R 75 ) (mean ± s.e., N = 4, n = 17, Student’s t-test, P = 0.47). Upper scale bar: 10 μm. Lower scale bar: 1 μm.

    Journal: Scientific Reports

    Article Title: STAC3 stably interacts through its C1 domain with CaV1.1 in skeletal muscle triads

    doi: 10.1038/srep41003

    Figure Lengend Snippet: STAC3 colocalizes also with heterologous Ca V 1.2 in skeletal muscle triads. ( A ) When co-expressed with Ca V 1.2, STAC3 is colocalized in clusters with the RyR and the Ca V subunits Ca V 1.2 and β 1a in dysgenic myotubes. Color overlay: 4X magnification of blue rectangle. Scale bars: 10 μm and 5 μm. ( B ) FRAP analysis of STAC3-GFP co-expressed with Ca V 1.2 shows very little recovery of fluorescence, very similar to when STAC3 is co-expressed with Ca V 1.1. Average recovery curves reveal similar low fluorescence recovery of STAC3-GFP co-expressed with Ca V 1.2 (red) and with Ca V 1.1 (blue) and comparable recovery rates 75 s after bleaching (R 75 ) (mean ± s.e., N = 4, n = 17, Student’s t-test, P = 0.47). Upper scale bar: 10 μm. Lower scale bar: 1 μm.

    Article Snippet: The primary antibodies rabbit serum anti-GFP (1:10,000; Invitrogen), mouse monoclonal anti-RyR (1:1000, cl.34 C, Alexis Biochemicals), mouse monoclonal anti-β1 (1:2000, cl.

    Techniques: Fluorescence

    Knockdown of human E-cadherin in MCF-7 cells and reconstitution with mouse EGFP-E-cadherin. (A) E-cadherin KD/E-cad-GFP reconstituted cell line: MCF-7 cells were infected with lentivirus bearing both shRNA directed against human E-cadherin and full-length mouse E-cadherin fused to EGFP. Immunoblots were probed for E-cadherin, Myosin IIA and IIB, and GAPDH. Protein levels of endogenous human E-cadherin (h) and expressed mouse Ecad-GFP (m) were recognized by the same E-cadherin antibody against the cytoplasmic tail. (B) E-cadherin KD/reconstituted MCF-7 cells were fixed and immunostained for human E-cadherin (magenta) to confirm knockdown and for EGFP (green) to visualize exogenous expression of E-cad-GFP. Cells were also costained for E-cad-GFP (green) and Myosin IIA (magenta) or Myosin IIB (magenta). Images shown are representative confocal sections from the apical junctions. (C) E-cadherin KD/reconstituted MCF-7 cells were infected with lentiviral shRNA directed against either Myosin IIA or Myosin IIB or with control empty virus expressing mCherry alone. Immunoblots show knockdown levels of Myosin IIA and IIB and expression levels of E-Cad-GFP. Tubulin was used as a loading control. (D) E-cadherin KD/reconstituted MCF-7 cells were infected with lentiviral shRNA directed against either Myosin IIA or Myosin IIB or with control empty virus expressing mCherry alone. Transduced cells express mCherry and are marked with asterisks. Fixed cells were immunostained for EGFP-E-cadherin and morphology of exogenously expressed E-cadherin in knockdown and control cells is shown at apical junctions. Arrows in enlarged images indicate accumulation of E-Cad-GFP at the ZA of cell-cell contacts. Scale bars = 10 µm.

    Journal: PLoS ONE

    Article Title: Multicomponent Analysis of Junctional Movements Regulated by Myosin II Isoforms at the Epithelial Zonula Adherens

    doi: 10.1371/journal.pone.0022458

    Figure Lengend Snippet: Knockdown of human E-cadherin in MCF-7 cells and reconstitution with mouse EGFP-E-cadherin. (A) E-cadherin KD/E-cad-GFP reconstituted cell line: MCF-7 cells were infected with lentivirus bearing both shRNA directed against human E-cadherin and full-length mouse E-cadherin fused to EGFP. Immunoblots were probed for E-cadherin, Myosin IIA and IIB, and GAPDH. Protein levels of endogenous human E-cadherin (h) and expressed mouse Ecad-GFP (m) were recognized by the same E-cadherin antibody against the cytoplasmic tail. (B) E-cadherin KD/reconstituted MCF-7 cells were fixed and immunostained for human E-cadherin (magenta) to confirm knockdown and for EGFP (green) to visualize exogenous expression of E-cad-GFP. Cells were also costained for E-cad-GFP (green) and Myosin IIA (magenta) or Myosin IIB (magenta). Images shown are representative confocal sections from the apical junctions. (C) E-cadherin KD/reconstituted MCF-7 cells were infected with lentiviral shRNA directed against either Myosin IIA or Myosin IIB or with control empty virus expressing mCherry alone. Immunoblots show knockdown levels of Myosin IIA and IIB and expression levels of E-Cad-GFP. Tubulin was used as a loading control. (D) E-cadherin KD/reconstituted MCF-7 cells were infected with lentiviral shRNA directed against either Myosin IIA or Myosin IIB or with control empty virus expressing mCherry alone. Transduced cells express mCherry and are marked with asterisks. Fixed cells were immunostained for EGFP-E-cadherin and morphology of exogenously expressed E-cadherin in knockdown and control cells is shown at apical junctions. Arrows in enlarged images indicate accumulation of E-Cad-GFP at the ZA of cell-cell contacts. Scale bars = 10 µm.

    Article Snippet: Antibodies Primary antibodies were as follows: (1) mouse mAb directed against the extracellular domain of E-cadherin (provided by Dr. M. Wheelock [University of Nebraska, Omaha, NE], with the permission of Dr. M. Takeichi [RIKEN CDB, Kobe, Japan]); (2) rabbit polyclonal antibody (pAb) for non-muscle myosin IIA heavy chain (Covance); (3) rabbit pAb for non-muscle myosin IIB heavy chain (Covance); (4) GFP- rabbit anti-GFP serum (Invitrogen); (5) E-cad- mouse monoclonal IgG2a against cytoplasmic tail (BD Transduction Laboratories); (5) GAPDH-rabbit polyclonal antibody (R & D systems). (6) mouse monoclonal Ab against β-tubulin (Sigma).

    Techniques: Infection, shRNA, Western Blot, Expressing

    Activation of UbDha in vivo a) In vivo labeling of endogenous E1 enzymes with Cy5-UbDha. Fluorescence scanning and immunoblotting of lysates from HeLa cell electroporated with the probe and harvested at indicated time intervals following electroporation with the probe. b) In vivo labeling of UBE1 with Cy5-UbDha following UBE1 inhibitor PYR-41 (50 µM) treatment. Fluorescence scan and quantification (% labeling in the absence of PYR-41; n=3, error bars correspond to SD, with significance (p) assessed using a two-sided t-test) are shown. c ) Distribution of Cy5-UbDha (magenta) in cells ectopically expressing GFP-UBE1 (green) relative to untransfected cells. Representative 3D confocal compilations of fixed cells treated as indicated are shown with DAPI (blue) overlays and nuclear insets; scale bars = 10 µm. d) Pixel traces of DAPI and Cy5-UbDha (marked with dotted lines in d) plotted as fluorescence over distance. e) .

    Journal: Nature chemical biology

    Article Title: A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

    doi: 10.1038/nchembio.2084

    Figure Lengend Snippet: Activation of UbDha in vivo a) In vivo labeling of endogenous E1 enzymes with Cy5-UbDha. Fluorescence scanning and immunoblotting of lysates from HeLa cell electroporated with the probe and harvested at indicated time intervals following electroporation with the probe. b) In vivo labeling of UBE1 with Cy5-UbDha following UBE1 inhibitor PYR-41 (50 µM) treatment. Fluorescence scan and quantification (% labeling in the absence of PYR-41; n=3, error bars correspond to SD, with significance (p) assessed using a two-sided t-test) are shown. c ) Distribution of Cy5-UbDha (magenta) in cells ectopically expressing GFP-UBE1 (green) relative to untransfected cells. Representative 3D confocal compilations of fixed cells treated as indicated are shown with DAPI (blue) overlays and nuclear insets; scale bars = 10 µm. d) Pixel traces of DAPI and Cy5-UbDha (marked with dotted lines in d) plotted as fluorescence over distance. e) .

    Article Snippet: Samples were resolved using standard SDS-PAGE, and probe reactivity was assessed by Cy5 fluorescence scanning (λex= 625 nm; λem= 680 nm), followed by gel transfer onto Nitrocellulose membranes and immunoblotting using either rabbit anti-GFP serum , mouse anti-Flag (1:1000 dilution; Sigma Aldrich, Sigma F3165) or mouse anti-β-actin (1:10000 dilution; Sigma A544), as indicated.

    Techniques: Activation Assay, In Vivo, Labeling, Fluorescence, Electroporation, Expressing

    Immunogold Localization of VHA-a1–GFP and TGN Morphology.

    Journal: The Plant Cell

    Article Title: Vacuolar H+-ATPase Activity Is Required for Endocytic and Secretory Trafficking in Arabidopsis [W]

    doi: 10.1105/tpc.105.037978

    Figure Lengend Snippet: Immunogold Localization of VHA-a1–GFP and TGN Morphology.

    Article Snippet: Immunogold labeling was performed on ultrathin thawed Tokuyasu cryosections of formaldehyde-fixed (8%, 3 h) and sucrose-infiltrated (2.1 M) root tips using rabbit anti-GFP serum (1:25; Abcam) or rabbit anti-VHA-E serum (1:500) ( ) and silver-enhanced (HQ Silver, 6 min; Nanoprobes) goat (Fab′) anti-rabbit IgG coupled to Nanogold (No. 2004; Nanoprobes).

    Techniques:

    Rapid Colocalization of VHA-a1–GFP with FM4-64.

    Journal: The Plant Cell

    Article Title: Vacuolar H+-ATPase Activity Is Required for Endocytic and Secretory Trafficking in Arabidopsis [W]

    doi: 10.1105/tpc.105.037978

    Figure Lengend Snippet: Rapid Colocalization of VHA-a1–GFP with FM4-64.

    Article Snippet: Immunogold labeling was performed on ultrathin thawed Tokuyasu cryosections of formaldehyde-fixed (8%, 3 h) and sucrose-infiltrated (2.1 M) root tips using rabbit anti-GFP serum (1:25; Abcam) or rabbit anti-VHA-E serum (1:500) ( ) and silver-enhanced (HQ Silver, 6 min; Nanoprobes) goat (Fab′) anti-rabbit IgG coupled to Nanogold (No. 2004; Nanoprobes).

    Techniques:

    Subcellular Localization of VHA-a–GFP Fusion Proteins Expressed in Roots of Seedlings.

    Journal: The Plant Cell

    Article Title: Vacuolar H+-ATPase Activity Is Required for Endocytic and Secretory Trafficking in Arabidopsis [W]

    doi: 10.1105/tpc.105.037978

    Figure Lengend Snippet: Subcellular Localization of VHA-a–GFP Fusion Proteins Expressed in Roots of Seedlings.

    Article Snippet: Immunogold labeling was performed on ultrathin thawed Tokuyasu cryosections of formaldehyde-fixed (8%, 3 h) and sucrose-infiltrated (2.1 M) root tips using rabbit anti-GFP serum (1:25; Abcam) or rabbit anti-VHA-E serum (1:500) ( ) and silver-enhanced (HQ Silver, 6 min; Nanoprobes) goat (Fab′) anti-rabbit IgG coupled to Nanogold (No. 2004; Nanoprobes).

    Techniques:

    Cleavage of wild-type (WT) and mutant HIV-1 Tat-GFP proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 polyclonal antibody as indicated.

    Journal: Journal of Virology

    Article Title: Human Immunodeficiency Virus Type 1 Protease Regulation of Tat Activity Is Essential for Efficient Reverse Transcription and Replication

    doi: 10.1128/JVI.77.18.9912-9921.2003

    Figure Lengend Snippet: Cleavage of wild-type (WT) and mutant HIV-1 Tat-GFP proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 polyclonal antibody as indicated.

    Article Snippet: Cell lysate samples were resolved on sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gels, and the proteins were transferred to polyvinylidene difluoride and probed with either human anti-HIV-1 immunoglobulin G (IgG) (National Institutes of Health [NIH] AIDS Research and Reference Reagent Program [ARRRP] catalog no. 192; diluted 1:6,000), HIV-1BH10 Tat monoclonal antibody (NIH ARRRP catalog no. 15.1), or anti-GFP rabbit serum (Molecular Probes catalog no. A6455; diluted 1:5,000).

    Techniques: Mutagenesis, Cleavage Assay, Labeling, Synthesized, Standard Deviation, Stable Transfection, DNA Synthesis, Transfection, Expressing, Polymerase Chain Reaction, Fluorescence, Western Blot, Infection, Purification

    Viral entry assays. A. HOG and HOG-PLP cells were infected with R120vGF at a m.o.i. of 1. After 24 h p.i., equal number of cells were subjected to SDS–PAGE and analyzed by immunoblotting with a rabbit polyclonal anti-HSV-1 antibody to detect immediate early proteins. In HOG-PLP cells, an increase in viral signal was observed. A positive control of HOG cells infected with HSV-1 was also included. The histogram corresponds to the quantification of the immunoblot signals expressed in arbitrary scanning units. B. Confluent monolayers of cells plated in 96-well tissue culture dishes were infected with a recombinant HSV-1 (KOS) gL86 at a m.o.i. of 10. After 6 h p.i., the β-galactosidase activity at 410 nm was analyzed in a microplate reader. Optical density (OD) was increased in HOG-PLP cells compared to HOG cells. C. To perform an antibody blocking assay, HOG cells blocked with an anti-PLP antibody were infected at a m.o.i. of 1 with K26GFP and processed for flow cytometry, analyzing fluorescence of GFP. Percentage (%) of max designates the number of cells relative to the maximum fraction. For each fluorescence intensity within positive cells, the percentage of cells incubated with blocking mouse anti-PLP antibody is considerably lower than control cells incubated without blocking antibodies (red plot) or cells incubated with a control mouse serum (yellow plot). Data are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells

    doi: 10.1371/journal.pone.0147885

    Figure Lengend Snippet: Viral entry assays. A. HOG and HOG-PLP cells were infected with R120vGF at a m.o.i. of 1. After 24 h p.i., equal number of cells were subjected to SDS–PAGE and analyzed by immunoblotting with a rabbit polyclonal anti-HSV-1 antibody to detect immediate early proteins. In HOG-PLP cells, an increase in viral signal was observed. A positive control of HOG cells infected with HSV-1 was also included. The histogram corresponds to the quantification of the immunoblot signals expressed in arbitrary scanning units. B. Confluent monolayers of cells plated in 96-well tissue culture dishes were infected with a recombinant HSV-1 (KOS) gL86 at a m.o.i. of 10. After 6 h p.i., the β-galactosidase activity at 410 nm was analyzed in a microplate reader. Optical density (OD) was increased in HOG-PLP cells compared to HOG cells. C. To perform an antibody blocking assay, HOG cells blocked with an anti-PLP antibody were infected at a m.o.i. of 1 with K26GFP and processed for flow cytometry, analyzing fluorescence of GFP. Percentage (%) of max designates the number of cells relative to the maximum fraction. For each fluorescence intensity within positive cells, the percentage of cells incubated with blocking mouse anti-PLP antibody is considerably lower than control cells incubated without blocking antibodies (red plot) or cells incubated with a control mouse serum (yellow plot). Data are representative of 3 independent experiments.

    Article Snippet: Anti-GFP rabbit polyclonal serum A6455, Alexa 488-, Alexa 647- and Alexa 594-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA).

    Techniques: Plasmid Purification, Infection, SDS Page, Positive Control, Recombinant, Activity Assay, Antibody Blocking Assay, Flow Cytometry, Cytometry, Fluorescence, Incubation, Blocking Assay

    Effect of PLP overexpression on HSV-1 infection. HOG and HOG-PLP cells were infected with HSV-1. PLP-transfected HOG cells showed higher susceptibility to HSV-1 than mock-transfected cells (A and B). A. Plaque assay showed an increase in the number of plaque forming units (p.f.u.) per ml in PLP-transfected cells compared to mock-transfected control cells. Two representative wells are also shown. The average plaque size of infected HOG-PLP cells is slightly larger than that of plaques in HOG cells. B. Cells were infected at a m.o.i. of 0.1 with HSV-1, and viral titers were determined 20 h p.i. by TCID 50 . Virus yield was significantly increased in PLP-transfected cells. The increment on viral yield correlated with the presence of PLP-EGFP, as shown by immunoblotting (C) with an anti-GFP antibody. C. HOG and HOG-PLP cells were infected with HSV-1 at an m.o.i. of 0,1. After 24 h p.i., equal number of cells were subjected to SDS–PAGE and analyzed by immunoblotting with a rabbit polyclonal anti-GFP antibody.

    Journal: PLoS ONE

    Article Title: Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells

    doi: 10.1371/journal.pone.0147885

    Figure Lengend Snippet: Effect of PLP overexpression on HSV-1 infection. HOG and HOG-PLP cells were infected with HSV-1. PLP-transfected HOG cells showed higher susceptibility to HSV-1 than mock-transfected cells (A and B). A. Plaque assay showed an increase in the number of plaque forming units (p.f.u.) per ml in PLP-transfected cells compared to mock-transfected control cells. Two representative wells are also shown. The average plaque size of infected HOG-PLP cells is slightly larger than that of plaques in HOG cells. B. Cells were infected at a m.o.i. of 0.1 with HSV-1, and viral titers were determined 20 h p.i. by TCID 50 . Virus yield was significantly increased in PLP-transfected cells. The increment on viral yield correlated with the presence of PLP-EGFP, as shown by immunoblotting (C) with an anti-GFP antibody. C. HOG and HOG-PLP cells were infected with HSV-1 at an m.o.i. of 0,1. After 24 h p.i., equal number of cells were subjected to SDS–PAGE and analyzed by immunoblotting with a rabbit polyclonal anti-GFP antibody.

    Article Snippet: Anti-GFP rabbit polyclonal serum A6455, Alexa 488-, Alexa 647- and Alexa 594-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA).

    Techniques: Plasmid Purification, Over Expression, Infection, Transfection, Plaque Assay, SDS Page

    Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of P[acman]-EGFP-dsh . Wings immunolabelled for GFP (green), or Stbm (red), and clones marked by loss of β-gal (blue).

    Journal: PLoS Genetics

    Article Title: DAnkrd49 and Bdbt act via Casein kinase Iε to regulate planar polarity in Drosophila

    doi: 10.1371/journal.pgen.1008820

    Figure Lengend Snippet: Loss of Dco activity reduces Dsh levels. (A) 28hr APF pupal wings carrying dco hypomorphic mutant clones of cells ( dco j3B9 ) , marked by loss of β-gal (blue). Wings immunolabelled for Dsh (green) or Fmi (red). Scale bar 10 μm. (B) 28hr APF pupal wings carrying clones of cells lacking dco ( dco j3B9 ), in a background expressing one copy of P[acman]-EGFP-dsh . Wings immunolabelled for GFP (green), or Stbm (red), and clones marked by loss of β-gal (blue).

    Article Snippet: Immunoprecipitations between Bdbt-EGFP and Myc-DAnkrd49 used GFP rabbit serum (ab290, Abcam), in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% TritonX-100, 1x protease inhibitor cocktail (#11697498001, Roche).

    Techniques: Activity Assay, Mutagenesis, Clone Assay, Expressing

    DAnkrd49 and Bdbt regulate Dsh levels. (A,B) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 ), marked by loss of GFP (red, A), or lacking Bdbt ( Bdbt Δ79 . 1 ), marked by loss of β-gal (red, B). Wings immunolabelled for Dsh (green). Scale bar 10 μm. (C-D) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 (C, DAnkrd49 l(2)35Be4 ), or clones of cells lacking Bdbt (D, Bdbt Δ79 . 1 ), in a background expressing one copy of P[acman]-EGFP-dsh . Wings immunolabelled for GFP (green), clones marked by loss of β-gal (red). (E) Representative western blot from wild-type pupal wings, or pupal wings expressing RNAi against DAnkrd49 (line 4140R-3 ), under control of the MS1096-GAL4 driver. Blot probed with Dsh (top) and Actin (bottom). Overall Dsh levels decrease in wings expressing DAnkrd49 RNAi. (F) Quantitation of Dsh levels from western blotting, normalised to Actin, from 6 biological replicates. Error bars are 95% confidence intervals, **p = 0.01 (unpaired t-test). See S2 Table for numerical data. (G) Comparison of levels of slow migrating Dsh (hyperphosphorylated) relative to fast migrating Dsh (less phosphorylated). The graph shows a ratio of the maximum intensity of the two bands averaged across the width of each band. Error bars are 95% confidence intervals, *p = 0.011 (unpaired t-test). See S2 Table for numerical data.

    Journal: PLoS Genetics

    Article Title: DAnkrd49 and Bdbt act via Casein kinase Iε to regulate planar polarity in Drosophila

    doi: 10.1371/journal.pgen.1008820

    Figure Lengend Snippet: DAnkrd49 and Bdbt regulate Dsh levels. (A,B) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 ), marked by loss of GFP (red, A), or lacking Bdbt ( Bdbt Δ79 . 1 ), marked by loss of β-gal (red, B). Wings immunolabelled for Dsh (green). Scale bar 10 μm. (C-D) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 (C, DAnkrd49 l(2)35Be4 ), or clones of cells lacking Bdbt (D, Bdbt Δ79 . 1 ), in a background expressing one copy of P[acman]-EGFP-dsh . Wings immunolabelled for GFP (green), clones marked by loss of β-gal (red). (E) Representative western blot from wild-type pupal wings, or pupal wings expressing RNAi against DAnkrd49 (line 4140R-3 ), under control of the MS1096-GAL4 driver. Blot probed with Dsh (top) and Actin (bottom). Overall Dsh levels decrease in wings expressing DAnkrd49 RNAi. (F) Quantitation of Dsh levels from western blotting, normalised to Actin, from 6 biological replicates. Error bars are 95% confidence intervals, **p = 0.01 (unpaired t-test). See S2 Table for numerical data. (G) Comparison of levels of slow migrating Dsh (hyperphosphorylated) relative to fast migrating Dsh (less phosphorylated). The graph shows a ratio of the maximum intensity of the two bands averaged across the width of each band. Error bars are 95% confidence intervals, *p = 0.011 (unpaired t-test). See S2 Table for numerical data.

    Article Snippet: Immunoprecipitations between Bdbt-EGFP and Myc-DAnkrd49 used GFP rabbit serum (ab290, Abcam), in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% TritonX-100, 1x protease inhibitor cocktail (#11697498001, Roche).

    Techniques: Clone Assay, Expressing, Western Blot, Quantitation Assay

    DAnkrd49 and Bdbt mutually regulate each other's protein levels. (A,B) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 ) in a background expressing ActP-DAnkrd49-EGFP (A), or clones of cells lacking Bdbt ( Bdbt Δ79 . 1 ) in a background expressing ActP-Bdbt-EGFP (B). Wings immunolabelled for GFP (green) and Stbm (red). Clones marked by loss of β-gal (blue). Note that levels of DAnkrd49-EGFP are slightly increased in the absence of endogenous DAnkrd49 . Scale bar 10 μm. (A',B') Clones in A and B showing the clone outline (yellow line) and the polarity nematic for each cell (red lines). Length of red line (polarity magnitude) is not significantly different in wild-type tissue and inside the DAnkrd49 and Bdbt clones, showing that the mutant phenotype is rescued by DAnkrd49-EGFP and Bdbt-EGFP respectively. (C,D) Quantitation of mean polarity and variation in polarity angle, in clones of DAnkrd49 in a background expressing ActP-DAnkrd49-EGFP (C) or clones of Bdbt in a background expressing ActP-Bdbt-EGFP (D), values from clone and non-clone regions of the same wing are linked by black bars. Paired t-tests were used to compare values in the same wing, no significant differences were found. 8 wings were quantified in (C) and 7 wings were quantified in (D). See S2 Table for numerical data. (E,F) 28hr APF pupal wings carrying clones of cells lacking Bdbt ( Bdbt Δ79 . 1 ) in a background expressing ActP-DAnkrd49-EGFP (E), or clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 ) in a background expressing ActP-Bdbt-EGFP (F). Wings immunolabelled for GFP (green) and Stbm (red). Clones marked by loss of β-gal (blue). Note that levels of the EGFP-tagged proteins are reduced within the clones.

    Journal: PLoS Genetics

    Article Title: DAnkrd49 and Bdbt act via Casein kinase Iε to regulate planar polarity in Drosophila

    doi: 10.1371/journal.pgen.1008820

    Figure Lengend Snippet: DAnkrd49 and Bdbt mutually regulate each other's protein levels. (A,B) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 ) in a background expressing ActP-DAnkrd49-EGFP (A), or clones of cells lacking Bdbt ( Bdbt Δ79 . 1 ) in a background expressing ActP-Bdbt-EGFP (B). Wings immunolabelled for GFP (green) and Stbm (red). Clones marked by loss of β-gal (blue). Note that levels of DAnkrd49-EGFP are slightly increased in the absence of endogenous DAnkrd49 . Scale bar 10 μm. (A',B') Clones in A and B showing the clone outline (yellow line) and the polarity nematic for each cell (red lines). Length of red line (polarity magnitude) is not significantly different in wild-type tissue and inside the DAnkrd49 and Bdbt clones, showing that the mutant phenotype is rescued by DAnkrd49-EGFP and Bdbt-EGFP respectively. (C,D) Quantitation of mean polarity and variation in polarity angle, in clones of DAnkrd49 in a background expressing ActP-DAnkrd49-EGFP (C) or clones of Bdbt in a background expressing ActP-Bdbt-EGFP (D), values from clone and non-clone regions of the same wing are linked by black bars. Paired t-tests were used to compare values in the same wing, no significant differences were found. 8 wings were quantified in (C) and 7 wings were quantified in (D). See S2 Table for numerical data. (E,F) 28hr APF pupal wings carrying clones of cells lacking Bdbt ( Bdbt Δ79 . 1 ) in a background expressing ActP-DAnkrd49-EGFP (E), or clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 ) in a background expressing ActP-Bdbt-EGFP (F). Wings immunolabelled for GFP (green) and Stbm (red). Clones marked by loss of β-gal (blue). Note that levels of the EGFP-tagged proteins are reduced within the clones.

    Article Snippet: Immunoprecipitations between Bdbt-EGFP and Myc-DAnkrd49 used GFP rabbit serum (ab290, Abcam), in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% TritonX-100, 1x protease inhibitor cocktail (#11697498001, Roche).

    Techniques: Clone Assay, Expressing, Mutagenesis, Quantitation Assay

    DAnkrd49 and Bdbt regulate asymmetric localisation of core proteins. (A,B) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 , A), or Bdbt ( Bdbt Δ79 . 1 , B), marked by loss of GFP or β-gal respectively (red). Wings immunolabelled for Fmi (green). Scale bar 10 μm. (A',B') Clones in A and B showing the clone outline (yellow line) and the polarity nematic for each cell (red lines). Length of red line (polarity magnitude) is reduced in DAnkrd49 and Bdbt clones compared to wild-type tissue. (C,D) Quantitation of mean polarity and variation in polarity angle, in wild-type and DAnkrd49 (C) or Bdbt (D) mutant tissue, values from the same wing are linked by black bars. Wild-type tissue was quantitated several cells away from clone boundaries, due to some disruption in polarity on clone boundaries. Paired t-tests were used to compare values in the same wing, **p

    Journal: PLoS Genetics

    Article Title: DAnkrd49 and Bdbt act via Casein kinase Iε to regulate planar polarity in Drosophila

    doi: 10.1371/journal.pgen.1008820

    Figure Lengend Snippet: DAnkrd49 and Bdbt regulate asymmetric localisation of core proteins. (A,B) 28hr APF pupal wings carrying clones of cells lacking DAnkrd49 ( DAnkrd49 l(2)35Be4 , A), or Bdbt ( Bdbt Δ79 . 1 , B), marked by loss of GFP or β-gal respectively (red). Wings immunolabelled for Fmi (green). Scale bar 10 μm. (A',B') Clones in A and B showing the clone outline (yellow line) and the polarity nematic for each cell (red lines). Length of red line (polarity magnitude) is reduced in DAnkrd49 and Bdbt clones compared to wild-type tissue. (C,D) Quantitation of mean polarity and variation in polarity angle, in wild-type and DAnkrd49 (C) or Bdbt (D) mutant tissue, values from the same wing are linked by black bars. Wild-type tissue was quantitated several cells away from clone boundaries, due to some disruption in polarity on clone boundaries. Paired t-tests were used to compare values in the same wing, **p

    Article Snippet: Immunoprecipitations between Bdbt-EGFP and Myc-DAnkrd49 used GFP rabbit serum (ab290, Abcam), in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% TritonX-100, 1x protease inhibitor cocktail (#11697498001, Roche).

    Techniques: Clone Assay, Quantitation Assay, Mutagenesis

    ParB spreads from parS (−1.6°) into comCDE . In the strains of interest, ParB was replaced by a ParB-GFP fusion. Exponentially growing cells were subjected to chromatin immunoprecipitation (ChIP) using anti-GFP antibodies, and the pulled-down DNA was subsequently analyzed by qPCR. (A and B) The WT (DLA42) versus the parS (−1.6°) mut strain (DLA43). (C and D) The parS (−1.6°) mut C strain (DLA77) versus the parS (−1.6°) mut NC strain (DLA80). The loci amplified by primer pairs 1, 2, 3, and 4 are shown in blue in Fig. 3A . The primer pair P amplifies another parS site [ parS (+2°)] situated +11 kb from oriC . Primer pairs T1 and T2 amplify 2 different loci in the terminus region. The graphs show pulldown efficiency (ChIP-DNA/input DNA × 100) for each primer pair. Note the different y axis scale between panels A and C and panels B and D.

    Journal: mBio

    Article Title: The ParB-parS Chromosome Segregation System Modulates Competence Development in Streptococcus pneumoniae

    doi: 10.1128/mBio.00662-15

    Figure Lengend Snippet: ParB spreads from parS (−1.6°) into comCDE . In the strains of interest, ParB was replaced by a ParB-GFP fusion. Exponentially growing cells were subjected to chromatin immunoprecipitation (ChIP) using anti-GFP antibodies, and the pulled-down DNA was subsequently analyzed by qPCR. (A and B) The WT (DLA42) versus the parS (−1.6°) mut strain (DLA43). (C and D) The parS (−1.6°) mut C strain (DLA77) versus the parS (−1.6°) mut NC strain (DLA80). The loci amplified by primer pairs 1, 2, 3, and 4 are shown in blue in Fig. 3A . The primer pair P amplifies another parS site [ parS (+2°)] situated +11 kb from oriC . Primer pairs T1 and T2 amplify 2 different loci in the terminus region. The graphs show pulldown efficiency (ChIP-DNA/input DNA × 100) for each primer pair. Note the different y axis scale between panels A and C and panels B and D.

    Article Snippet: Cells were washed and sonicated, and then immunoprecipitation was performed using anti-GFP antibodies (rabbit serum, polyclonal; Invitrogen A-6455) and protein G-coupled Dynabeads (Invitrogen).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification