anti gfp mouse monoclonal antibody  (Cell Signaling Technology Inc)


Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc anti gfp mouse monoclonal antibody
    ( A ) Scheme for expression of Cp OGA WT or Cp OGA DM in various Drosophila brain structures using different Gal4 drivers. ( B ) Immunostaining of adult Drosophila brains. Brains were stained with anti- O- GlcNAc antibody RL2 (red) to assess O- GlcNAcylation level, <t>and</t> <t>anti-GFP</t> (green) antibody to validate tissue-specific expression of Cp OGA. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( C ) Quantification of fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brains. n = 4. ( D ) Immunostaining of adult Drosophila brains. Outlined areas indicate the cell bodies of Kenyon cells in mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brain structures. n = 8. ( F ) A compilation of performance index in learning test of the indicated flies expressing either Cp OGA WT or Cp OGA DM . n = 6-8. ( G ) A compilation of learning performance index of flies expressing Cp OGA WT or Cp OGA DM only in the mushroom body at adult stage. n = 6. Each datapoint represents an independent experiment with approximately 200 flies. p- values were determined by unpaired t -test, and the stars indicate significant differences (***p<0.001, **p<0.01 and ns, not significant, p≥0.05). Error bars represent SD. Figure 1—source data 1. Excel spreadsheet containing source data used to generate .
    Anti Gfp Mouse Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gfp mouse monoclonal antibody/product/Cell Signaling Technology Inc
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    anti gfp mouse monoclonal antibody - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Tissue-specific O- GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning"

    Article Title: Tissue-specific O- GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

    Journal: eLife

    doi: 10.7554/eLife.91269

    ( A ) Scheme for expression of Cp OGA WT or Cp OGA DM in various Drosophila brain structures using different Gal4 drivers. ( B ) Immunostaining of adult Drosophila brains. Brains were stained with anti- O- GlcNAc antibody RL2 (red) to assess O- GlcNAcylation level, and anti-GFP (green) antibody to validate tissue-specific expression of Cp OGA. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( C ) Quantification of fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brains. n = 4. ( D ) Immunostaining of adult Drosophila brains. Outlined areas indicate the cell bodies of Kenyon cells in mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brain structures. n = 8. ( F ) A compilation of performance index in learning test of the indicated flies expressing either Cp OGA WT or Cp OGA DM . n = 6-8. ( G ) A compilation of learning performance index of flies expressing Cp OGA WT or Cp OGA DM only in the mushroom body at adult stage. n = 6. Each datapoint represents an independent experiment with approximately 200 flies. p- values were determined by unpaired t -test, and the stars indicate significant differences (***p<0.001, **p<0.01 and ns, not significant, p≥0.05). Error bars represent SD. Figure 1—source data 1. Excel spreadsheet containing source data used to generate .
    Figure Legend Snippet: ( A ) Scheme for expression of Cp OGA WT or Cp OGA DM in various Drosophila brain structures using different Gal4 drivers. ( B ) Immunostaining of adult Drosophila brains. Brains were stained with anti- O- GlcNAc antibody RL2 (red) to assess O- GlcNAcylation level, and anti-GFP (green) antibody to validate tissue-specific expression of Cp OGA. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( C ) Quantification of fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brains. n = 4. ( D ) Immunostaining of adult Drosophila brains. Outlined areas indicate the cell bodies of Kenyon cells in mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brain structures. n = 8. ( F ) A compilation of performance index in learning test of the indicated flies expressing either Cp OGA WT or Cp OGA DM . n = 6-8. ( G ) A compilation of learning performance index of flies expressing Cp OGA WT or Cp OGA DM only in the mushroom body at adult stage. n = 6. Each datapoint represents an independent experiment with approximately 200 flies. p- values were determined by unpaired t -test, and the stars indicate significant differences (***p<0.001, **p<0.01 and ns, not significant, p≥0.05). Error bars represent SD. Figure 1—source data 1. Excel spreadsheet containing source data used to generate .

    Techniques Used: Expressing, Immunostaining, Staining

    ( A ) Heatmaps showing the mRNA levels (upper) and the normalized O- GlcNAc levels (lower) of the identified ribosomal candidates in different brain regions. ( B ) Immunoprecipitation of ribosomes using FLAG-tagged RpL13A. The expression of RpL13A-FLAG was validated by immunoblotting with anti-FLAG antibody. Ribosomal proteins were visualized using silver staining, and O- GlcNAcylation of ribosomes was analyzed by immunoblotting with anti- O- GlcNAc antibody RL2. ( C ) A compilation of the performance index of the indicated flies in the learning test. Learning defect of flies expressing Cp OGA WT was corrected by selective expression of dMyc in mushroom body. n = 6-7. Each datapoint represents an independent experiment with approximately 200 flies. ( D ) Ex vivo measurement of protein synthesis in mushroom body using the O-propargyl-puromycin (OPP) assay. Brains from the indicated flies were stained with anti-GFP (green) antibody to validate Cp OGA expression, and OPP (gray) to quantify protein synthesis. Nuclei were visualized with DAPI (blue). Outlined areas indicate the cell bodies of Kenyon cells of mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative OPP fluorescent intensity in mushroom body regions. n = 8-12. p -values were determined by unpaired t -test, the stars indicate significant differences (***p<0.001, **p<0.01, *p<0.05). Error bars represent SD. Figure 4—source data 1. Raw data of all western blots for . Figure 4—source data 2. Complete and uncropped membranes of all western blots for . Figure 4—source data 3. Excel spreadsheet containing source data used to generate .
    Figure Legend Snippet: ( A ) Heatmaps showing the mRNA levels (upper) and the normalized O- GlcNAc levels (lower) of the identified ribosomal candidates in different brain regions. ( B ) Immunoprecipitation of ribosomes using FLAG-tagged RpL13A. The expression of RpL13A-FLAG was validated by immunoblotting with anti-FLAG antibody. Ribosomal proteins were visualized using silver staining, and O- GlcNAcylation of ribosomes was analyzed by immunoblotting with anti- O- GlcNAc antibody RL2. ( C ) A compilation of the performance index of the indicated flies in the learning test. Learning defect of flies expressing Cp OGA WT was corrected by selective expression of dMyc in mushroom body. n = 6-7. Each datapoint represents an independent experiment with approximately 200 flies. ( D ) Ex vivo measurement of protein synthesis in mushroom body using the O-propargyl-puromycin (OPP) assay. Brains from the indicated flies were stained with anti-GFP (green) antibody to validate Cp OGA expression, and OPP (gray) to quantify protein synthesis. Nuclei were visualized with DAPI (blue). Outlined areas indicate the cell bodies of Kenyon cells of mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative OPP fluorescent intensity in mushroom body regions. n = 8-12. p -values were determined by unpaired t -test, the stars indicate significant differences (***p<0.001, **p<0.01, *p<0.05). Error bars represent SD. Figure 4—source data 1. Raw data of all western blots for . Figure 4—source data 2. Complete and uncropped membranes of all western blots for . Figure 4—source data 3. Excel spreadsheet containing source data used to generate .

    Techniques Used: Immunoprecipitation, Expressing, Western Blot, Silver Staining, Ex Vivo, Staining


    Figure Legend Snippet:

    Techniques Used: Silver Staining, Protease Inhibitor, Magnetic Beads, SYBR Green Assay, Sequencing, Modification, Software, Microscopy

    gfp mouse monoclonal primary antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc gfp mouse monoclonal primary antibody
    ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F <t>)</t> <t>Kir7.1</t> expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. <t>GFP</t> primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.
    Gfp Mouse Monoclonal Primary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy"

    Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI171356

    ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F ) Kir7.1 expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. GFP primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.
    Figure Legend Snippet: ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F ) Kir7.1 expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. GFP primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.

    Techniques Used: Construct, Generated, Mutagenesis, Sequencing, Electroporation, Expressing, Immunocytochemistry, Staining, Membrane

    gfp mouse monoclonal primary antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc gfp mouse monoclonal primary antibody
    Gfp Mouse Monoclonal Primary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp mouse monoclonal primary antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    gfp mouse monoclonal primary antibody - by Bioz Stars, 2024-06
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    mouse monoclonal anti gfp  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc mouse monoclonal anti gfp
    (A) Schematic of Mup1 sorting. (B) <t>Mup1-GFP</t> localization after methionine stimulation. (C) Western blotting analysis of Mup1 sorting. Cell lysates were analyzed by immunoblotting <t>using</t> <t>anti-GFP</t> and anti-G6PDH antibodies. Vacuole delivery of Mup1-GFP yields protease-resistant GFP fragments (GFP’). (D) Quantification of Mup1-GFP processing. (E) GFP-CPS localization. (F) Quantification of GFP-CPS localization of each category. (G) Schematic of retromer-mediated Vps10 recycling. (H) Vps10-GFP localization in WT, vps35 Δ (retromer), and vps13 Δ cells. (I) Quantification of Vps10-GFP localization. Scale bar: 1 µm.
    Mouse Monoclonal Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse monoclonal anti gfp - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting"

    Article Title: A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

    Journal: bioRxiv

    doi: 10.1101/2023.07.06.547997

    (A) Schematic of Mup1 sorting. (B) Mup1-GFP localization after methionine stimulation. (C) Western blotting analysis of Mup1 sorting. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. Vacuole delivery of Mup1-GFP yields protease-resistant GFP fragments (GFP’). (D) Quantification of Mup1-GFP processing. (E) GFP-CPS localization. (F) Quantification of GFP-CPS localization of each category. (G) Schematic of retromer-mediated Vps10 recycling. (H) Vps10-GFP localization in WT, vps35 Δ (retromer), and vps13 Δ cells. (I) Quantification of Vps10-GFP localization. Scale bar: 1 µm.
    Figure Legend Snippet: (A) Schematic of Mup1 sorting. (B) Mup1-GFP localization after methionine stimulation. (C) Western blotting analysis of Mup1 sorting. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. Vacuole delivery of Mup1-GFP yields protease-resistant GFP fragments (GFP’). (D) Quantification of Mup1-GFP processing. (E) GFP-CPS localization. (F) Quantification of GFP-CPS localization of each category. (G) Schematic of retromer-mediated Vps10 recycling. (H) Vps10-GFP localization in WT, vps35 Δ (retromer), and vps13 Δ cells. (I) Quantification of Vps10-GFP localization. Scale bar: 1 µm.

    Techniques Used: Western Blot

    (A) Localization of Vps13-GFP. Scale bar: 1 µm. (B) Quantification of Vps13-GFP colocalizing with mCherry-Vps21. (C) Localization of Vps13 ΔC -GFP (residues 1-1851). Scale bar: 1 µm. (D) Quantification of Vps13-GFP puncta localization. (E) The localization of Vps13 N -GFP (residues 1-39). Scale bar: 1 µm. (F) Line scan analysis for the region highlighted by the yellow dash line in E. (G) Vps13-GFP localization at the ER-endosome contact site. Vps13-GFP, DsRed-HDEL (ER), and Mup1-BFP (endosome) expressing cells were stimulated with methionine. Scale bar: 1 µm. (H) Live cell-imaging analysis of the ER (GFP-HDEL) and endosome (mCherry-Vps21). Scale bar: 500 nm. (I) ER diameters per 100 nm interval starting at the endosome contact along 500 nm length of ER. (J and K) Mup1-GFP processing in vps13 mutants after methionine stimulation. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies.
    Figure Legend Snippet: (A) Localization of Vps13-GFP. Scale bar: 1 µm. (B) Quantification of Vps13-GFP colocalizing with mCherry-Vps21. (C) Localization of Vps13 ΔC -GFP (residues 1-1851). Scale bar: 1 µm. (D) Quantification of Vps13-GFP puncta localization. (E) The localization of Vps13 N -GFP (residues 1-39). Scale bar: 1 µm. (F) Line scan analysis for the region highlighted by the yellow dash line in E. (G) Vps13-GFP localization at the ER-endosome contact site. Vps13-GFP, DsRed-HDEL (ER), and Mup1-BFP (endosome) expressing cells were stimulated with methionine. Scale bar: 1 µm. (H) Live cell-imaging analysis of the ER (GFP-HDEL) and endosome (mCherry-Vps21). Scale bar: 500 nm. (I) ER diameters per 100 nm interval starting at the endosome contact along 500 nm length of ER. (J and K) Mup1-GFP processing in vps13 mutants after methionine stimulation. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies.

    Techniques Used: Expressing, Live Cell Imaging, Western Blot

    (A) Schematic of ESCRT-mediated sorting at the endosome. (B) Can1-FKBP processing after rapamycin treatments. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. (C) GFP-Vps27 (ESCRT-0) localization. (D) Snf7-GFP (ESCRT-III) localization. Scale bar: 1 µm.
    Figure Legend Snippet: (A) Schematic of ESCRT-mediated sorting at the endosome. (B) Can1-FKBP processing after rapamycin treatments. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. (C) GFP-Vps27 (ESCRT-0) localization. (D) Snf7-GFP (ESCRT-III) localization. Scale bar: 1 µm.

    Techniques Used: Western Blot

    (A) The ubiquitination of Mup1-GFP. Mup1-GFP expressing cells were stimulated with methionine for 15 min and then immunoprecipitated under denatured conditions. The immunoprecipitated (IP) products were analyzed by immunoblotting using anti-GFP and anti-ubiquitin antibodies. (B) Schematic of a rapamycin-dependent degradation system for Can1. (C) Can1-FKBP localization. Rapamycin-insensitive mutant cells ( tor1-1 , fpr1 Δ) expressing Can1-GFP-2xFKBP (Can1-FKBP) and FRB-3xUb were treated with rapamycin. (D) Quantification of Can-FKBP sorting. (E) Vps4-GFP localization. (F) Quantification of endosomal Vps4-GFP localization. (G) Schematic of Mup1-pHluorin-mCherry sorting. (H) Mup1-pHluorin-mCherry localization. Mup1-pHluorin-mCherry expressing vam7 ts and vam7 t vps13 Δ cells were incubated at 37°C and stimulated with methionine. (I) pHluorin fluorescence of Mup1-pHluorin-mCherry. Scale bar: 1 µm.
    Figure Legend Snippet: (A) The ubiquitination of Mup1-GFP. Mup1-GFP expressing cells were stimulated with methionine for 15 min and then immunoprecipitated under denatured conditions. The immunoprecipitated (IP) products were analyzed by immunoblotting using anti-GFP and anti-ubiquitin antibodies. (B) Schematic of a rapamycin-dependent degradation system for Can1. (C) Can1-FKBP localization. Rapamycin-insensitive mutant cells ( tor1-1 , fpr1 Δ) expressing Can1-GFP-2xFKBP (Can1-FKBP) and FRB-3xUb were treated with rapamycin. (D) Quantification of Can-FKBP sorting. (E) Vps4-GFP localization. (F) Quantification of endosomal Vps4-GFP localization. (G) Schematic of Mup1-pHluorin-mCherry sorting. (H) Mup1-pHluorin-mCherry localization. Mup1-pHluorin-mCherry expressing vam7 ts and vam7 t vps13 Δ cells were incubated at 37°C and stimulated with methionine. (I) pHluorin fluorescence of Mup1-pHluorin-mCherry. Scale bar: 1 µm.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Incubation, Fluorescence

    mouse monoclonal anti gfp  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc mouse monoclonal anti gfp
    Mouse Monoclonal Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti gfp/product/Cell Signaling Technology Inc
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    mouse anti β actin  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc mouse anti β actin
    (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and <t>β-actin</t> was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).
    Mouse Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti β actin - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff"

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    Journal: bioRxiv

    doi: 10.1101/2023.03.20.533404

    (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and β-actin was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).
    Figure Legend Snippet: (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and β-actin was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Sequencing, Standard Deviation, Synthesized, Immunofluorescence, Fluorescence

    (A) Plasmid encoding Flag-tagged catalytic-deficient nsp15 H223A, H238A, oligomerization-deficient nsp15 D285A, D315A, wild type nsp15, and the vector PXJ40, were each co-transfected with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP into DF-1 cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands was analysed by Image J, normalized to the signal of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 transfected cells. (B) DF-1 (C) Vero and H1299 cells, were transfected with the plasmid encoding wild type or mutated nsp15 and treated with puromycin (5 µg/ml) for 1 h at 23 h post-transfection (h.p.t), to label the de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated in the right panel (histogram plot).
    Figure Legend Snippet: (A) Plasmid encoding Flag-tagged catalytic-deficient nsp15 H223A, H238A, oligomerization-deficient nsp15 D285A, D315A, wild type nsp15, and the vector PXJ40, were each co-transfected with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP into DF-1 cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands was analysed by Image J, normalized to the signal of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 transfected cells. (B) DF-1 (C) Vero and H1299 cells, were transfected with the plasmid encoding wild type or mutated nsp15 and treated with puromycin (5 µg/ml) for 1 h at 23 h post-transfection (h.p.t), to label the de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated in the right panel (histogram plot).

    Techniques Used: Plasmid Preparation, Transfection, Western Blot, Synthesized, Immunofluorescence, Fluorescence

    The plasmid encoding wild type or catalytic-deficient nsp15 from the indicated coronaviruses was co-transfected with the plasmid encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands of EGFP or IBV N were analysed by Image J, normalized to the signal of β-actin and presented relative to the PXJ40 group.
    Figure Legend Snippet: The plasmid encoding wild type or catalytic-deficient nsp15 from the indicated coronaviruses was co-transfected with the plasmid encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands of EGFP or IBV N were analysed by Image J, normalized to the signal of β-actin and presented relative to the PXJ40 group.

    Techniques Used: Plasmid Preparation, Transfection, Western Blot

    Plasmid encoding wild type or catalytic-deficient IBV nsp15 and reporter plasmid encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40.
    Figure Legend Snippet: Plasmid encoding wild type or catalytic-deficient IBV nsp15 and reporter plasmid encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40.

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

    (A) DF-1 cells or (B) H1299 cells were infected with IBV-WT or rIBV-nsp15H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/ml) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N protein, and β-actin. Density of the puromycin labelled proteins was normalized to the signal of β-actin. Ratio of the puromycin–labelled de novo peptides of the infected cells (+) to that of the uninfected cells (-) at the same time h.p.i. is shown. (B) H1299 cells were infected as described above followed by dot blot analysis to detects dsRNA and western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α.
    Figure Legend Snippet: (A) DF-1 cells or (B) H1299 cells were infected with IBV-WT or rIBV-nsp15H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/ml) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N protein, and β-actin. Density of the puromycin labelled proteins was normalized to the signal of β-actin. Ratio of the puromycin–labelled de novo peptides of the infected cells (+) to that of the uninfected cells (-) at the same time h.p.i. is shown. (B) H1299 cells were infected as described above followed by dot blot analysis to detects dsRNA and western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α.

    Techniques Used: Infection, Western Blot, Dot Blot

    (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).
    Figure Legend Snippet: (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).

    Techniques Used: Transfection, Plasmid Preparation, Immunofluorescence, FLAG-tag, Staining, Infection, Western Blot

    mouse anti gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti gfp
    Mouse Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti gfp monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti gfp monoclonal antibody
    (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence <t>of</t> <t>PBS</t> (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with <t>GFP</t> tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.
    Mouse Anti Gfp Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production"

    Article Title: Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041624

    (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence of PBS (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with GFP tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.
    Figure Legend Snippet: (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence of PBS (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with GFP tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.

    Techniques Used: Infection, Negative Control, Positive Control, Fluorescence

    mouse monoclonal anti gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse monoclonal anti gfp
    (A) Expression plasmids were transfected into HEK293 cells for 24 h. Cell lysates were used to detect the fusion genes’ expression by Western blot using antibodies as indicated. (B) Flag-LC3 or Flag-LC3 A120 was transfected into HEK293 cells for 24 h. 50 µM of Chloroquine (CQ) was used to induce Flag- LC3-II accumulation for 2 h before harvesting the cells. Flag-LC3 is normally lipidated as demonstrated by Flag-LC3-II accumulation after CQ treatment. No such accumulation was seen in Flag-LC3 A120 mutant after CQ treatment demonstrating that this mutant does not pass through the lipidation process. (C) MEFwt cells were transfected with indicated expression plasmids for 24 h before they were recorded by fluorescence <t>microscopy.</t> <t>GFP-Atg4B,</t> GFP-Atg7 and GFP-Atg3 were mainly distributed in the cytosol. No puncta was observed at any given time. Scale bar = 25micron.
    Mouse Monoclonal Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I"

    Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073229

    (A) Expression plasmids were transfected into HEK293 cells for 24 h. Cell lysates were used to detect the fusion genes’ expression by Western blot using antibodies as indicated. (B) Flag-LC3 or Flag-LC3 A120 was transfected into HEK293 cells for 24 h. 50 µM of Chloroquine (CQ) was used to induce Flag- LC3-II accumulation for 2 h before harvesting the cells. Flag-LC3 is normally lipidated as demonstrated by Flag-LC3-II accumulation after CQ treatment. No such accumulation was seen in Flag-LC3 A120 mutant after CQ treatment demonstrating that this mutant does not pass through the lipidation process. (C) MEFwt cells were transfected with indicated expression plasmids for 24 h before they were recorded by fluorescence microscopy. GFP-Atg4B, GFP-Atg7 and GFP-Atg3 were mainly distributed in the cytosol. No puncta was observed at any given time. Scale bar = 25micron.
    Figure Legend Snippet: (A) Expression plasmids were transfected into HEK293 cells for 24 h. Cell lysates were used to detect the fusion genes’ expression by Western blot using antibodies as indicated. (B) Flag-LC3 or Flag-LC3 A120 was transfected into HEK293 cells for 24 h. 50 µM of Chloroquine (CQ) was used to induce Flag- LC3-II accumulation for 2 h before harvesting the cells. Flag-LC3 is normally lipidated as demonstrated by Flag-LC3-II accumulation after CQ treatment. No such accumulation was seen in Flag-LC3 A120 mutant after CQ treatment demonstrating that this mutant does not pass through the lipidation process. (C) MEFwt cells were transfected with indicated expression plasmids for 24 h before they were recorded by fluorescence microscopy. GFP-Atg4B, GFP-Atg7 and GFP-Atg3 were mainly distributed in the cytosol. No puncta was observed at any given time. Scale bar = 25micron.

    Techniques Used: Expressing, Transfection, Western Blot, Mutagenesis, Fluorescence, Microscopy

    GFP-Atg4B and mRFP-p62 expression plasmids (A) or with Flag-LC3 (B) were co-transfected into MEFwt cells for 24h. GFP-Atg4B C74A and mRFP-p62 with Flag-LC3 vectors (C) or with Flag-LC3 A120 vector (D) were co-transfected into MEFwt cells for 24 h. GFP-Atg4B or GFP-Atg4B C74A expression vector was co-transfected with Flag-LC3 (E) or Flag-LC3 A120 (F) into HEK293 cells for 48 h. Immunoprecipitation (IP) was performed using total cell lysates. Immunoblotting was conducted using the antibodies as indicated. (G) mRFP-p62, GFP-Atg4B C74A and Flag-LC3 expression vectors were co-transfected into MEFwt cells for 24 h. Colocalization of Flag-LC3 within mRFP-p62 aggregates were detected by immunofluorescence staining (IF) using Flag M2 antibody. Fluorescence images presented in this study were representative of at least three independent experiments. Scale bar = 25micron.
    Figure Legend Snippet: GFP-Atg4B and mRFP-p62 expression plasmids (A) or with Flag-LC3 (B) were co-transfected into MEFwt cells for 24h. GFP-Atg4B C74A and mRFP-p62 with Flag-LC3 vectors (C) or with Flag-LC3 A120 vector (D) were co-transfected into MEFwt cells for 24 h. GFP-Atg4B or GFP-Atg4B C74A expression vector was co-transfected with Flag-LC3 (E) or Flag-LC3 A120 (F) into HEK293 cells for 48 h. Immunoprecipitation (IP) was performed using total cell lysates. Immunoblotting was conducted using the antibodies as indicated. (G) mRFP-p62, GFP-Atg4B C74A and Flag-LC3 expression vectors were co-transfected into MEFwt cells for 24 h. Colocalization of Flag-LC3 within mRFP-p62 aggregates were detected by immunofluorescence staining (IF) using Flag M2 antibody. Fluorescence images presented in this study were representative of at least three independent experiments. Scale bar = 25micron.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Fluorescence

    Vectors expressing mRFP-p62, GFP-LC3, or GFP-LC3 A120 were transfected into MEFatg5 −/− cells as indicated (A and B). Images were taken at 48 h time point. The same sets of plasmids were transfected into MEFwt cells as indicated for 72 h. Images were recorded at 20 h and 72 h post transfection (C–F). Scale bar = 25micro. Arrows point to mRFP-p62 only punctae. The inserts were enlarged to show the puncta in detail.
    Figure Legend Snippet: Vectors expressing mRFP-p62, GFP-LC3, or GFP-LC3 A120 were transfected into MEFatg5 −/− cells as indicated (A and B). Images were taken at 48 h time point. The same sets of plasmids were transfected into MEFwt cells as indicated for 72 h. Images were recorded at 20 h and 72 h post transfection (C–F). Scale bar = 25micro. Arrows point to mRFP-p62 only punctae. The inserts were enlarged to show the puncta in detail.

    Techniques Used: Expressing, Transfection

    GFP-Atg7 (Aa), GFP-Atg7/Flag-LC3 (Ab), or GFP-Atg7 C572A /Flag-LC3 (Ac) vectors were co-transfected with mRFP-p62 into MEFwt cells for 48 h. GFP-Atg7 punctae were shown; mRFP-p62 and Flag-LC3 with GFP-Atg7 vectors (B) or with GFP-Atg7 C572A (C) were co-transfected into MEFwt cells for 48 h; mRFP-p62 and Flag-LC3A 120 with GFP-Atg7 vectors (D) or with GFP-Atg7 C572A (E) were co-transfected into MEFwt cells for 48 h. Flag-LC3, GFP-Atg7, or GFP-Atg7 C572A expression vectors were co-transfected into HEK293 cells as indicated for 48 h. Total cell lysates were used for IP by the Flag M2 antibody. Immunoblotting was conducted using the antibodies as indicated (F and G). Expressing vectors of GFP-Atg12 and mRFP-p62 were co-transfected into MEFwt cells for 48 h (H). All images were recorded at 48 h post-transfection. Scale bar = 25 micron. The inserts were enlarged to show the puncta in detail.
    Figure Legend Snippet: GFP-Atg7 (Aa), GFP-Atg7/Flag-LC3 (Ab), or GFP-Atg7 C572A /Flag-LC3 (Ac) vectors were co-transfected with mRFP-p62 into MEFwt cells for 48 h. GFP-Atg7 punctae were shown; mRFP-p62 and Flag-LC3 with GFP-Atg7 vectors (B) or with GFP-Atg7 C572A (C) were co-transfected into MEFwt cells for 48 h; mRFP-p62 and Flag-LC3A 120 with GFP-Atg7 vectors (D) or with GFP-Atg7 C572A (E) were co-transfected into MEFwt cells for 48 h. Flag-LC3, GFP-Atg7, or GFP-Atg7 C572A expression vectors were co-transfected into HEK293 cells as indicated for 48 h. Total cell lysates were used for IP by the Flag M2 antibody. Immunoblotting was conducted using the antibodies as indicated (F and G). Expressing vectors of GFP-Atg12 and mRFP-p62 were co-transfected into MEFwt cells for 48 h (H). All images were recorded at 48 h post-transfection. Scale bar = 25 micron. The inserts were enlarged to show the puncta in detail.

    Techniques Used: Transfection, Expressing, Western Blot

    GFP-Atg3 and mRFP-p62 (A) or with Flag-LC3 vectors (B) were co-transfected into MEFwt cells for 48 h. GFP-Atg3 C264S , mRFP-p62 and Flag-LC3 vectors were co-transfected into MEFwt cells for 48 h (C). Flag-LC3, GFP-Atg3, GFP-Atg3 C264S , or GFP-Atg3 C264A expression vectors were co-transfected into HEK293 cells for 48 h. Immunoprecipitation was conducted using total cell lysates. Immunoblotting was conducted as indicated. Images were recorded at 48 h post-transfection. Scale bar = 25 micron.
    Figure Legend Snippet: GFP-Atg3 and mRFP-p62 (A) or with Flag-LC3 vectors (B) were co-transfected into MEFwt cells for 48 h. GFP-Atg3 C264S , mRFP-p62 and Flag-LC3 vectors were co-transfected into MEFwt cells for 48 h (C). Flag-LC3, GFP-Atg3, GFP-Atg3 C264S , or GFP-Atg3 C264A expression vectors were co-transfected into HEK293 cells for 48 h. Immunoprecipitation was conducted using total cell lysates. Immunoblotting was conducted as indicated. Images were recorded at 48 h post-transfection. Scale bar = 25 micron.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot

    HEK293 cells were co-transfected with mRFP-p62/GFP-Atg3/Flag-LC3 or mRFP-p62/GFP-Atg4B/Flag-LC3 for 48 h. Total cell lysates were used to conduct IP using correspondent antibodies. Immunoblottings were then applied using antibodies as indicated (A and B). HEK293 cells were transfected with GFP-Atg4B or GFP-Atg4B C74A alone or with mRFP-p62 for 48 h (C). HEK293 cells were transfected with GFP-Atg3 alone or with mRFP-p62 for 48 h (D). Total cell lysates were used to detect LC3 species as indicated. Relative band intensity of protein of interest was divided by correspondent band intensity of β-actin loading control. Fold of increase or decrease was then determined by setting the control sample as 1.
    Figure Legend Snippet: HEK293 cells were co-transfected with mRFP-p62/GFP-Atg3/Flag-LC3 or mRFP-p62/GFP-Atg4B/Flag-LC3 for 48 h. Total cell lysates were used to conduct IP using correspondent antibodies. Immunoblottings were then applied using antibodies as indicated (A and B). HEK293 cells were transfected with GFP-Atg4B or GFP-Atg4B C74A alone or with mRFP-p62 for 48 h (C). HEK293 cells were transfected with GFP-Atg3 alone or with mRFP-p62 for 48 h (D). Total cell lysates were used to detect LC3 species as indicated. Relative band intensity of protein of interest was divided by correspondent band intensity of β-actin loading control. Fold of increase or decrease was then determined by setting the control sample as 1.

    Techniques Used: Transfection

    HEK293-GFP-LC3 cells were cultured in nutrient-rich media (Aa) or treated with CPP for 2 h (Ab) to induce large GFP-LC3 positive vesicles. HEK293-GFP-LC3 A120 cells were used as a control (Ac); (B) HEK293-GFP-LC3 cells were transfected with mRFP-p62 for 24 h, and then treated with CPP for 2 h to induce large GFP-LC3/mRFP-p62 positive vesicles. (C) HEK293-GFP-LC3 cells were transfected with mRFP-p62-LRS-mut.2 for 24 h, and then treated with CPP for 2 h. HEK293-GFP-LC3 cell line was cultured in regular media (D) or treated with CPP for 2 h (E) before fixed and immunostained for the endogenous LC3 and p62. (F) HEK293-GFP-LC3 cells were cultured in regular media or treated with CPP for 2 h. Total cell lysates were used for immunoprecipitation of endogenous p62. Western blot analysis was conducted to detect p62 bound GFP-LC3 species. Arrow points to GFP-LC3 positive vesicle. Arrow head points to p62 aggregate. Scale bar = 25 micron.
    Figure Legend Snippet: HEK293-GFP-LC3 cells were cultured in nutrient-rich media (Aa) or treated with CPP for 2 h (Ab) to induce large GFP-LC3 positive vesicles. HEK293-GFP-LC3 A120 cells were used as a control (Ac); (B) HEK293-GFP-LC3 cells were transfected with mRFP-p62 for 24 h, and then treated with CPP for 2 h to induce large GFP-LC3/mRFP-p62 positive vesicles. (C) HEK293-GFP-LC3 cells were transfected with mRFP-p62-LRS-mut.2 for 24 h, and then treated with CPP for 2 h. HEK293-GFP-LC3 cell line was cultured in regular media (D) or treated with CPP for 2 h (E) before fixed and immunostained for the endogenous LC3 and p62. (F) HEK293-GFP-LC3 cells were cultured in regular media or treated with CPP for 2 h. Total cell lysates were used for immunoprecipitation of endogenous p62. Western blot analysis was conducted to detect p62 bound GFP-LC3 species. Arrow points to GFP-LC3 positive vesicle. Arrow head points to p62 aggregate. Scale bar = 25 micron.

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

    monoclonal mouse antibody against gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc monoclonal mouse antibody against gfp
    Monoclonal Mouse Antibody Against Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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  • 86
    Cell Signaling Technology Inc anti gfp mouse monoclonal antibody
    ( A ) Scheme for expression of Cp OGA WT or Cp OGA DM in various Drosophila brain structures using different Gal4 drivers. ( B ) Immunostaining of adult Drosophila brains. Brains were stained with anti- O- GlcNAc antibody RL2 (red) to assess O- GlcNAcylation level, <t>and</t> <t>anti-GFP</t> (green) antibody to validate tissue-specific expression of Cp OGA. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( C ) Quantification of fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brains. n = 4. ( D ) Immunostaining of adult Drosophila brains. Outlined areas indicate the cell bodies of Kenyon cells in mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brain structures. n = 8. ( F ) A compilation of performance index in learning test of the indicated flies expressing either Cp OGA WT or Cp OGA DM . n = 6-8. ( G ) A compilation of learning performance index of flies expressing Cp OGA WT or Cp OGA DM only in the mushroom body at adult stage. n = 6. Each datapoint represents an independent experiment with approximately 200 flies. p- values were determined by unpaired t -test, and the stars indicate significant differences (***p<0.001, **p<0.01 and ns, not significant, p≥0.05). Error bars represent SD. Figure 1—source data 1. Excel spreadsheet containing source data used to generate .
    Anti Gfp Mouse Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gfp mouse monoclonal primary antibody
    ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F <t>)</t> <t>Kir7.1</t> expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. <t>GFP</t> primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.
    Gfp Mouse Monoclonal Primary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc mouse monoclonal anti gfp
    (A) Schematic of Mup1 sorting. (B) <t>Mup1-GFP</t> localization after methionine stimulation. (C) Western blotting analysis of Mup1 sorting. Cell lysates were analyzed by immunoblotting <t>using</t> <t>anti-GFP</t> and anti-G6PDH antibodies. Vacuole delivery of Mup1-GFP yields protease-resistant GFP fragments (GFP’). (D) Quantification of Mup1-GFP processing. (E) GFP-CPS localization. (F) Quantification of GFP-CPS localization of each category. (G) Schematic of retromer-mediated Vps10 recycling. (H) Vps10-GFP localization in WT, vps35 Δ (retromer), and vps13 Δ cells. (I) Quantification of Vps10-GFP localization. Scale bar: 1 µm.
    Mouse Monoclonal Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc mouse anti β actin
    (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and <t>β-actin</t> was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).
    Mouse Anti β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc mouse anti gfp
    (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and <t>β-actin</t> was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).
    Mouse Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti gfp/product/Cell Signaling Technology Inc
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    94
    Cell Signaling Technology Inc mouse anti gfp monoclonal antibody
    (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence <t>of</t> <t>PBS</t> (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with <t>GFP</t> tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.
    Mouse Anti Gfp Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti gfp monoclonal antibody/product/Cell Signaling Technology Inc
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    Cell Signaling Technology Inc monoclonal mouse antibody against gfp
    (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence <t>of</t> <t>PBS</t> (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with <t>GFP</t> tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.
    Monoclonal Mouse Antibody Against Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal mouse antibody against gfp/product/Cell Signaling Technology Inc
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    Image Search Results


    ( A ) Scheme for expression of Cp OGA WT or Cp OGA DM in various Drosophila brain structures using different Gal4 drivers. ( B ) Immunostaining of adult Drosophila brains. Brains were stained with anti- O- GlcNAc antibody RL2 (red) to assess O- GlcNAcylation level, and anti-GFP (green) antibody to validate tissue-specific expression of Cp OGA. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( C ) Quantification of fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brains. n = 4. ( D ) Immunostaining of adult Drosophila brains. Outlined areas indicate the cell bodies of Kenyon cells in mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brain structures. n = 8. ( F ) A compilation of performance index in learning test of the indicated flies expressing either Cp OGA WT or Cp OGA DM . n = 6-8. ( G ) A compilation of learning performance index of flies expressing Cp OGA WT or Cp OGA DM only in the mushroom body at adult stage. n = 6. Each datapoint represents an independent experiment with approximately 200 flies. p- values were determined by unpaired t -test, and the stars indicate significant differences (***p<0.001, **p<0.01 and ns, not significant, p≥0.05). Error bars represent SD. Figure 1—source data 1. Excel spreadsheet containing source data used to generate .

    Journal: eLife

    Article Title: Tissue-specific O- GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

    doi: 10.7554/eLife.91269

    Figure Lengend Snippet: ( A ) Scheme for expression of Cp OGA WT or Cp OGA DM in various Drosophila brain structures using different Gal4 drivers. ( B ) Immunostaining of adult Drosophila brains. Brains were stained with anti- O- GlcNAc antibody RL2 (red) to assess O- GlcNAcylation level, and anti-GFP (green) antibody to validate tissue-specific expression of Cp OGA. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. ( C ) Quantification of fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brains. n = 4. ( D ) Immunostaining of adult Drosophila brains. Outlined areas indicate the cell bodies of Kenyon cells in mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative fluorescent intensity of O- GlcNAc staining in Cp OGA WT or Cp OGA DM expressed brain structures. n = 8. ( F ) A compilation of performance index in learning test of the indicated flies expressing either Cp OGA WT or Cp OGA DM . n = 6-8. ( G ) A compilation of learning performance index of flies expressing Cp OGA WT or Cp OGA DM only in the mushroom body at adult stage. n = 6. Each datapoint represents an independent experiment with approximately 200 flies. p- values were determined by unpaired t -test, and the stars indicate significant differences (***p<0.001, **p<0.01 and ns, not significant, p≥0.05). Error bars represent SD. Figure 1—source data 1. Excel spreadsheet containing source data used to generate .

    Article Snippet: Antibody , Anti-GFP Mouse Monoclonal Antibody (4B10) , Cell Signaling Technology , Cat# 2955, RRID: AB_1196614 , WB (1:1000) IF (1:200).

    Techniques: Expressing, Immunostaining, Staining

    ( A ) Heatmaps showing the mRNA levels (upper) and the normalized O- GlcNAc levels (lower) of the identified ribosomal candidates in different brain regions. ( B ) Immunoprecipitation of ribosomes using FLAG-tagged RpL13A. The expression of RpL13A-FLAG was validated by immunoblotting with anti-FLAG antibody. Ribosomal proteins were visualized using silver staining, and O- GlcNAcylation of ribosomes was analyzed by immunoblotting with anti- O- GlcNAc antibody RL2. ( C ) A compilation of the performance index of the indicated flies in the learning test. Learning defect of flies expressing Cp OGA WT was corrected by selective expression of dMyc in mushroom body. n = 6-7. Each datapoint represents an independent experiment with approximately 200 flies. ( D ) Ex vivo measurement of protein synthesis in mushroom body using the O-propargyl-puromycin (OPP) assay. Brains from the indicated flies were stained with anti-GFP (green) antibody to validate Cp OGA expression, and OPP (gray) to quantify protein synthesis. Nuclei were visualized with DAPI (blue). Outlined areas indicate the cell bodies of Kenyon cells of mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative OPP fluorescent intensity in mushroom body regions. n = 8-12. p -values were determined by unpaired t -test, the stars indicate significant differences (***p<0.001, **p<0.01, *p<0.05). Error bars represent SD. Figure 4—source data 1. Raw data of all western blots for . Figure 4—source data 2. Complete and uncropped membranes of all western blots for . Figure 4—source data 3. Excel spreadsheet containing source data used to generate .

    Journal: eLife

    Article Title: Tissue-specific O- GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

    doi: 10.7554/eLife.91269

    Figure Lengend Snippet: ( A ) Heatmaps showing the mRNA levels (upper) and the normalized O- GlcNAc levels (lower) of the identified ribosomal candidates in different brain regions. ( B ) Immunoprecipitation of ribosomes using FLAG-tagged RpL13A. The expression of RpL13A-FLAG was validated by immunoblotting with anti-FLAG antibody. Ribosomal proteins were visualized using silver staining, and O- GlcNAcylation of ribosomes was analyzed by immunoblotting with anti- O- GlcNAc antibody RL2. ( C ) A compilation of the performance index of the indicated flies in the learning test. Learning defect of flies expressing Cp OGA WT was corrected by selective expression of dMyc in mushroom body. n = 6-7. Each datapoint represents an independent experiment with approximately 200 flies. ( D ) Ex vivo measurement of protein synthesis in mushroom body using the O-propargyl-puromycin (OPP) assay. Brains from the indicated flies were stained with anti-GFP (green) antibody to validate Cp OGA expression, and OPP (gray) to quantify protein synthesis. Nuclei were visualized with DAPI (blue). Outlined areas indicate the cell bodies of Kenyon cells of mushroom body. Scale bar: 100 μm. ( E ) Quantification of relative OPP fluorescent intensity in mushroom body regions. n = 8-12. p -values were determined by unpaired t -test, the stars indicate significant differences (***p<0.001, **p<0.01, *p<0.05). Error bars represent SD. Figure 4—source data 1. Raw data of all western blots for . Figure 4—source data 2. Complete and uncropped membranes of all western blots for . Figure 4—source data 3. Excel spreadsheet containing source data used to generate .

    Article Snippet: Antibody , Anti-GFP Mouse Monoclonal Antibody (4B10) , Cell Signaling Technology , Cat# 2955, RRID: AB_1196614 , WB (1:1000) IF (1:200).

    Techniques: Immunoprecipitation, Expressing, Western Blot, Silver Staining, Ex Vivo, Staining

    Journal: eLife

    Article Title: Tissue-specific O- GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

    doi: 10.7554/eLife.91269

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-GFP Mouse Monoclonal Antibody (4B10) , Cell Signaling Technology , Cat# 2955, RRID: AB_1196614 , WB (1:1000) IF (1:200).

    Techniques: Silver Staining, Protease Inhibitor, Magnetic Beads, SYBR Green Assay, Sequencing, Modification, Software, Microscopy

    ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F ) Kir7.1 expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. GFP primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.

    Journal: The Journal of Clinical Investigation

    Article Title: Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

    doi: 10.1172/JCI171356

    Figure Lengend Snippet: ( A ) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. ( B ) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).( C ) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). ( D ) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293 W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( E ) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells ( n = 3). Markers (diamonds) represent the individual biological replicates ( n = 3), and error bars represent SEM by 2-tailed Student’s t test. ( F ) Kir7.1 expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. GFP primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.

    Article Snippet: As the protein is GFP tagged, GFP mouse monoclonal primary antibody (Cell Signaling, 2955, 1:250) was used to enhance the Kir7.1 protein expression for its detection in the cells.

    Techniques: Construct, Generated, Mutagenesis, Sequencing, Electroporation, Expressing, Immunocytochemistry, Staining, Membrane

    (A) Schematic of Mup1 sorting. (B) Mup1-GFP localization after methionine stimulation. (C) Western blotting analysis of Mup1 sorting. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. Vacuole delivery of Mup1-GFP yields protease-resistant GFP fragments (GFP’). (D) Quantification of Mup1-GFP processing. (E) GFP-CPS localization. (F) Quantification of GFP-CPS localization of each category. (G) Schematic of retromer-mediated Vps10 recycling. (H) Vps10-GFP localization in WT, vps35 Δ (retromer), and vps13 Δ cells. (I) Quantification of Vps10-GFP localization. Scale bar: 1 µm.

    Journal: bioRxiv

    Article Title: A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

    doi: 10.1101/2023.07.06.547997

    Figure Lengend Snippet: (A) Schematic of Mup1 sorting. (B) Mup1-GFP localization after methionine stimulation. (C) Western blotting analysis of Mup1 sorting. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. Vacuole delivery of Mup1-GFP yields protease-resistant GFP fragments (GFP’). (D) Quantification of Mup1-GFP processing. (E) GFP-CPS localization. (F) Quantification of GFP-CPS localization of each category. (G) Schematic of retromer-mediated Vps10 recycling. (H) Vps10-GFP localization in WT, vps35 Δ (retromer), and vps13 Δ cells. (I) Quantification of Vps10-GFP localization. Scale bar: 1 µm.

    Article Snippet: For immunoblotting, mouse monoclonal anti-GFP (B-2; Santa Cruz, Sc-9996), rabbit polyclonal anti-G6PDH (Sigma-Aldrich, SAB2100871), and anti-ubiquitin (P4D1; Cell Signaling, #3936) were used at dilution factors of 1:5000, 1:10,000 and 1:1000, respectively.

    Techniques: Western Blot

    (A) Localization of Vps13-GFP. Scale bar: 1 µm. (B) Quantification of Vps13-GFP colocalizing with mCherry-Vps21. (C) Localization of Vps13 ΔC -GFP (residues 1-1851). Scale bar: 1 µm. (D) Quantification of Vps13-GFP puncta localization. (E) The localization of Vps13 N -GFP (residues 1-39). Scale bar: 1 µm. (F) Line scan analysis for the region highlighted by the yellow dash line in E. (G) Vps13-GFP localization at the ER-endosome contact site. Vps13-GFP, DsRed-HDEL (ER), and Mup1-BFP (endosome) expressing cells were stimulated with methionine. Scale bar: 1 µm. (H) Live cell-imaging analysis of the ER (GFP-HDEL) and endosome (mCherry-Vps21). Scale bar: 500 nm. (I) ER diameters per 100 nm interval starting at the endosome contact along 500 nm length of ER. (J and K) Mup1-GFP processing in vps13 mutants after methionine stimulation. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies.

    Journal: bioRxiv

    Article Title: A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

    doi: 10.1101/2023.07.06.547997

    Figure Lengend Snippet: (A) Localization of Vps13-GFP. Scale bar: 1 µm. (B) Quantification of Vps13-GFP colocalizing with mCherry-Vps21. (C) Localization of Vps13 ΔC -GFP (residues 1-1851). Scale bar: 1 µm. (D) Quantification of Vps13-GFP puncta localization. (E) The localization of Vps13 N -GFP (residues 1-39). Scale bar: 1 µm. (F) Line scan analysis for the region highlighted by the yellow dash line in E. (G) Vps13-GFP localization at the ER-endosome contact site. Vps13-GFP, DsRed-HDEL (ER), and Mup1-BFP (endosome) expressing cells were stimulated with methionine. Scale bar: 1 µm. (H) Live cell-imaging analysis of the ER (GFP-HDEL) and endosome (mCherry-Vps21). Scale bar: 500 nm. (I) ER diameters per 100 nm interval starting at the endosome contact along 500 nm length of ER. (J and K) Mup1-GFP processing in vps13 mutants after methionine stimulation. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies.

    Article Snippet: For immunoblotting, mouse monoclonal anti-GFP (B-2; Santa Cruz, Sc-9996), rabbit polyclonal anti-G6PDH (Sigma-Aldrich, SAB2100871), and anti-ubiquitin (P4D1; Cell Signaling, #3936) were used at dilution factors of 1:5000, 1:10,000 and 1:1000, respectively.

    Techniques: Expressing, Live Cell Imaging, Western Blot

    (A) Schematic of ESCRT-mediated sorting at the endosome. (B) Can1-FKBP processing after rapamycin treatments. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. (C) GFP-Vps27 (ESCRT-0) localization. (D) Snf7-GFP (ESCRT-III) localization. Scale bar: 1 µm.

    Journal: bioRxiv

    Article Title: A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

    doi: 10.1101/2023.07.06.547997

    Figure Lengend Snippet: (A) Schematic of ESCRT-mediated sorting at the endosome. (B) Can1-FKBP processing after rapamycin treatments. Cell lysates were analyzed by immunoblotting using anti-GFP and anti-G6PDH antibodies. (C) GFP-Vps27 (ESCRT-0) localization. (D) Snf7-GFP (ESCRT-III) localization. Scale bar: 1 µm.

    Article Snippet: For immunoblotting, mouse monoclonal anti-GFP (B-2; Santa Cruz, Sc-9996), rabbit polyclonal anti-G6PDH (Sigma-Aldrich, SAB2100871), and anti-ubiquitin (P4D1; Cell Signaling, #3936) were used at dilution factors of 1:5000, 1:10,000 and 1:1000, respectively.

    Techniques: Western Blot

    (A) The ubiquitination of Mup1-GFP. Mup1-GFP expressing cells were stimulated with methionine for 15 min and then immunoprecipitated under denatured conditions. The immunoprecipitated (IP) products were analyzed by immunoblotting using anti-GFP and anti-ubiquitin antibodies. (B) Schematic of a rapamycin-dependent degradation system for Can1. (C) Can1-FKBP localization. Rapamycin-insensitive mutant cells ( tor1-1 , fpr1 Δ) expressing Can1-GFP-2xFKBP (Can1-FKBP) and FRB-3xUb were treated with rapamycin. (D) Quantification of Can-FKBP sorting. (E) Vps4-GFP localization. (F) Quantification of endosomal Vps4-GFP localization. (G) Schematic of Mup1-pHluorin-mCherry sorting. (H) Mup1-pHluorin-mCherry localization. Mup1-pHluorin-mCherry expressing vam7 ts and vam7 t vps13 Δ cells were incubated at 37°C and stimulated with methionine. (I) pHluorin fluorescence of Mup1-pHluorin-mCherry. Scale bar: 1 µm.

    Journal: bioRxiv

    Article Title: A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

    doi: 10.1101/2023.07.06.547997

    Figure Lengend Snippet: (A) The ubiquitination of Mup1-GFP. Mup1-GFP expressing cells were stimulated with methionine for 15 min and then immunoprecipitated under denatured conditions. The immunoprecipitated (IP) products were analyzed by immunoblotting using anti-GFP and anti-ubiquitin antibodies. (B) Schematic of a rapamycin-dependent degradation system for Can1. (C) Can1-FKBP localization. Rapamycin-insensitive mutant cells ( tor1-1 , fpr1 Δ) expressing Can1-GFP-2xFKBP (Can1-FKBP) and FRB-3xUb were treated with rapamycin. (D) Quantification of Can-FKBP sorting. (E) Vps4-GFP localization. (F) Quantification of endosomal Vps4-GFP localization. (G) Schematic of Mup1-pHluorin-mCherry sorting. (H) Mup1-pHluorin-mCherry localization. Mup1-pHluorin-mCherry expressing vam7 ts and vam7 t vps13 Δ cells were incubated at 37°C and stimulated with methionine. (I) pHluorin fluorescence of Mup1-pHluorin-mCherry. Scale bar: 1 µm.

    Article Snippet: For immunoblotting, mouse monoclonal anti-GFP (B-2; Santa Cruz, Sc-9996), rabbit polyclonal anti-G6PDH (Sigma-Aldrich, SAB2100871), and anti-ubiquitin (P4D1; Cell Signaling, #3936) were used at dilution factors of 1:5000, 1:10,000 and 1:1000, respectively.

    Techniques: Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Incubation, Fluorescence

    (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and β-actin was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).

    Journal: bioRxiv

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    doi: 10.1101/2023.03.20.533404

    Figure Lengend Snippet: (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and β-actin was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).

    Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Sequencing, Standard Deviation, Synthesized, Immunofluorescence, Fluorescence

    (A) Plasmid encoding Flag-tagged catalytic-deficient nsp15 H223A, H238A, oligomerization-deficient nsp15 D285A, D315A, wild type nsp15, and the vector PXJ40, were each co-transfected with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP into DF-1 cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands was analysed by Image J, normalized to the signal of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 transfected cells. (B) DF-1 (C) Vero and H1299 cells, were transfected with the plasmid encoding wild type or mutated nsp15 and treated with puromycin (5 µg/ml) for 1 h at 23 h post-transfection (h.p.t), to label the de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated in the right panel (histogram plot).

    Journal: bioRxiv

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    doi: 10.1101/2023.03.20.533404

    Figure Lengend Snippet: (A) Plasmid encoding Flag-tagged catalytic-deficient nsp15 H223A, H238A, oligomerization-deficient nsp15 D285A, D315A, wild type nsp15, and the vector PXJ40, were each co-transfected with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP into DF-1 cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands was analysed by Image J, normalized to the signal of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 transfected cells. (B) DF-1 (C) Vero and H1299 cells, were transfected with the plasmid encoding wild type or mutated nsp15 and treated with puromycin (5 µg/ml) for 1 h at 23 h post-transfection (h.p.t), to label the de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated in the right panel (histogram plot).

    Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

    Techniques: Plasmid Preparation, Transfection, Western Blot, Synthesized, Immunofluorescence, Fluorescence

    The plasmid encoding wild type or catalytic-deficient nsp15 from the indicated coronaviruses was co-transfected with the plasmid encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands of EGFP or IBV N were analysed by Image J, normalized to the signal of β-actin and presented relative to the PXJ40 group.

    Journal: bioRxiv

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    doi: 10.1101/2023.03.20.533404

    Figure Lengend Snippet: The plasmid encoding wild type or catalytic-deficient nsp15 from the indicated coronaviruses was co-transfected with the plasmid encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands of EGFP or IBV N were analysed by Image J, normalized to the signal of β-actin and presented relative to the PXJ40 group.

    Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

    Techniques: Plasmid Preparation, Transfection, Western Blot

    Plasmid encoding wild type or catalytic-deficient IBV nsp15 and reporter plasmid encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40.

    Journal: bioRxiv

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    doi: 10.1101/2023.03.20.533404

    Figure Lengend Snippet: Plasmid encoding wild type or catalytic-deficient IBV nsp15 and reporter plasmid encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40.

    Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

    Techniques: Plasmid Preparation, Luciferase, Incubation, Western Blot, Transfection

    (A) DF-1 cells or (B) H1299 cells were infected with IBV-WT or rIBV-nsp15H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/ml) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N protein, and β-actin. Density of the puromycin labelled proteins was normalized to the signal of β-actin. Ratio of the puromycin–labelled de novo peptides of the infected cells (+) to that of the uninfected cells (-) at the same time h.p.i. is shown. (B) H1299 cells were infected as described above followed by dot blot analysis to detects dsRNA and western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α.

    Journal: bioRxiv

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    doi: 10.1101/2023.03.20.533404

    Figure Lengend Snippet: (A) DF-1 cells or (B) H1299 cells were infected with IBV-WT or rIBV-nsp15H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/ml) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N protein, and β-actin. Density of the puromycin labelled proteins was normalized to the signal of β-actin. Ratio of the puromycin–labelled de novo peptides of the infected cells (+) to that of the uninfected cells (-) at the same time h.p.i. is shown. (B) H1299 cells were infected as described above followed by dot blot analysis to detects dsRNA and western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α.

    Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

    Techniques: Infection, Western Blot, Dot Blot

    (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).

    Journal: bioRxiv

    Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

    doi: 10.1101/2023.03.20.533404

    Figure Lengend Snippet: (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).

    Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

    Techniques: Transfection, Plasmid Preparation, Immunofluorescence, FLAG-tag, Staining, Infection, Western Blot

    (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence of PBS (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with GFP tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.

    Journal: PLoS ONE

    Article Title: Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production

    doi: 10.1371/journal.pone.0041624

    Figure Lengend Snippet: (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence of PBS (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with GFP tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.

    Article Snippet: Subsequently, the fixed cells were washed three times with PBS and incubated with mouse anti-GFP monoclonal antibody (Cell Signaling Technology, Beverly, MA) diluted 1∶200 in PBS with 1% BSA for 1 h at room temperature.

    Techniques: Infection, Negative Control, Positive Control, Fluorescence