laci 6xhis pra fusion protein  (Thermo Fisher)


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    Structured Review

    Thermo Fisher laci 6xhis pra fusion protein
    Purification of minichromosomes (MiniCs) from highly synchronous meiosis. a Structure of MiniCs. Recombination hotspot ( M26 ) and basal control ( BC ) MiniCs contain different alleles of the ade6 gene, a fission yeast origin of replication ( ARS ) and copies of the LacO DNA site for affinity purification. b Efficiency and synchrony of induced meiosis. Plots show the frequencies of cells undergoing the first meiotic division ( MI , 2 nuclei) and having completed the second meiotic division ( MII , 3–4 nuclei) in strains harboring the indicated MiniCs. c The indicated samples of chromatin from steps of purification were deproteinized, and their DNAs were analyzed by agarose gel electrophoresis ( WCE , whole-cell extract). d MiniC copy number and degree of enrichment; note log scale. The abundance of ade6 DNA in the chromosome ( Chr ) or in the MiniC (with chromosomal ade6 deleted) was determined by qPCR, relative to the act1 locus, and those values were normalized relative to single-copy ade6 in the chromosome. Affinity purifications ( AP ) employed <t>LacI-6xHis-prA;</t> mock AP samples were processed identically, but lacked LacI-6xHis-prA. In this figure and others, plots with error bars are mean ± SD from three or more biological replicates
    Laci 6xhis Pra Fusion Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot"

    Article Title: Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0233-x

    Purification of minichromosomes (MiniCs) from highly synchronous meiosis. a Structure of MiniCs. Recombination hotspot ( M26 ) and basal control ( BC ) MiniCs contain different alleles of the ade6 gene, a fission yeast origin of replication ( ARS ) and copies of the LacO DNA site for affinity purification. b Efficiency and synchrony of induced meiosis. Plots show the frequencies of cells undergoing the first meiotic division ( MI , 2 nuclei) and having completed the second meiotic division ( MII , 3–4 nuclei) in strains harboring the indicated MiniCs. c The indicated samples of chromatin from steps of purification were deproteinized, and their DNAs were analyzed by agarose gel electrophoresis ( WCE , whole-cell extract). d MiniC copy number and degree of enrichment; note log scale. The abundance of ade6 DNA in the chromosome ( Chr ) or in the MiniC (with chromosomal ade6 deleted) was determined by qPCR, relative to the act1 locus, and those values were normalized relative to single-copy ade6 in the chromosome. Affinity purifications ( AP ) employed LacI-6xHis-prA; mock AP samples were processed identically, but lacked LacI-6xHis-prA. In this figure and others, plots with error bars are mean ± SD from three or more biological replicates
    Figure Legend Snippet: Purification of minichromosomes (MiniCs) from highly synchronous meiosis. a Structure of MiniCs. Recombination hotspot ( M26 ) and basal control ( BC ) MiniCs contain different alleles of the ade6 gene, a fission yeast origin of replication ( ARS ) and copies of the LacO DNA site for affinity purification. b Efficiency and synchrony of induced meiosis. Plots show the frequencies of cells undergoing the first meiotic division ( MI , 2 nuclei) and having completed the second meiotic division ( MII , 3–4 nuclei) in strains harboring the indicated MiniCs. c The indicated samples of chromatin from steps of purification were deproteinized, and their DNAs were analyzed by agarose gel electrophoresis ( WCE , whole-cell extract). d MiniC copy number and degree of enrichment; note log scale. The abundance of ade6 DNA in the chromosome ( Chr ) or in the MiniC (with chromosomal ade6 deleted) was determined by qPCR, relative to the act1 locus, and those values were normalized relative to single-copy ade6 in the chromosome. Affinity purifications ( AP ) employed LacI-6xHis-prA; mock AP samples were processed identically, but lacked LacI-6xHis-prA. In this figure and others, plots with error bars are mean ± SD from three or more biological replicates

    Techniques Used: Purification, Affinity Purification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    2) Product Images from "A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3"

    Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

    Journal: mAbs

    doi: 10.1080/19420862.2018.1445451

    Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.
    Figure Legend Snippet: Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.

    Techniques Used: Expressing, Construct, Modification, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Isolation, Negative Control, Purification, Incubation, Generated, SDS Page, Labeling, Fluorescence, Confocal Microscopy, Binding Assay

    Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.
    Figure Legend Snippet: Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Generated, Activity Assay, Binding Assay

    3) Product Images from "Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries"

    Article Title: Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3814-3

    Recombinant peptide chip optimization. a Assessment of best E. coli growth intervals for consistent chip performance. A series of E. coli cells transformed with either Strep-tag constructs or empty vector were imprinted on nitrocellulose, grown thereon for the specified period, then exposed to IPTG, and subsequently lysed. Released peptides were analysed for their interaction with HRP-labeled Streptavidin. b Signal intensities (and so a rough estimation of affinities) between Strep-tagII and Dim12G were evaluated in relation to empty vector control, as indicated. Different peptide spot numbers per single standardized microtiter plate well are compared. c Consistent pin performance and maximal detection sensitivity were assessed by spotting a dilution series of biotinylated test protein. d Steps required for recombinant peptide chip assembly
    Figure Legend Snippet: Recombinant peptide chip optimization. a Assessment of best E. coli growth intervals for consistent chip performance. A series of E. coli cells transformed with either Strep-tag constructs or empty vector were imprinted on nitrocellulose, grown thereon for the specified period, then exposed to IPTG, and subsequently lysed. Released peptides were analysed for their interaction with HRP-labeled Streptavidin. b Signal intensities (and so a rough estimation of affinities) between Strep-tagII and Dim12G were evaluated in relation to empty vector control, as indicated. Different peptide spot numbers per single standardized microtiter plate well are compared. c Consistent pin performance and maximal detection sensitivity were assessed by spotting a dilution series of biotinylated test protein. d Steps required for recombinant peptide chip assembly

    Techniques Used: Recombinant, Chromatin Immunoprecipitation, Transformation Assay, Strep-tag, Construct, Plasmid Preparation, Labeling

    4) Product Images from "Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling"

    Article Title: Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

    Journal: bioRxiv

    doi: 10.1101/632547

    A . Flowchart for ProP-PD screen, motif enrichment and experimental validation of CN-Binding peptides. B . CN Sequence read counts for selected peptide-phage using WT or mutant calcineurin over 4 selection rounds are shown. Experiments were carried out in buffer containing CaCl 2 or EDTA as indicated. CN NIR contains 330 NIR 332 -AAA mutations; CN WF contains 352 W-A, 356 F-A mutations in CNAα. Phage selections were robust for CN WT and CN NIR but not for CN WF . C . Distribution of read counts for the indicated peptide type between CN WT and CN NIR . D . In vitro binding of purified His-tagged CN WT , CN NIR and CN WF to GST-tagged LxVP peptide from NFATC1 or a high affinity PxIxIT peptide, VIVIT ( Aramburu et al., 1999 ). CN NIR has substantially reduced binding to VIVIT but not to NFATC1-LxVP whereas CN WF has reduced binding to NFATC1-LXVP but not to VIVIT. See also Table S1.
    Figure Legend Snippet: A . Flowchart for ProP-PD screen, motif enrichment and experimental validation of CN-Binding peptides. B . CN Sequence read counts for selected peptide-phage using WT or mutant calcineurin over 4 selection rounds are shown. Experiments were carried out in buffer containing CaCl 2 or EDTA as indicated. CN NIR contains 330 NIR 332 -AAA mutations; CN WF contains 352 W-A, 356 F-A mutations in CNAα. Phage selections were robust for CN WT and CN NIR but not for CN WF . C . Distribution of read counts for the indicated peptide type between CN WT and CN NIR . D . In vitro binding of purified His-tagged CN WT , CN NIR and CN WF to GST-tagged LxVP peptide from NFATC1 or a high affinity PxIxIT peptide, VIVIT ( Aramburu et al., 1999 ). CN NIR has substantially reduced binding to VIVIT but not to NFATC1-LxVP whereas CN WF has reduced binding to NFATC1-LXVP but not to VIVIT. See also Table S1.

    Techniques Used: Binding Assay, Sequencing, Mutagenesis, Selection, In Vitro, Purification

    5) Product Images from "Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization"

    Article Title: Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0491-9

    Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.
    Figure Legend Snippet: Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.

    Techniques Used: Agglutination, Negative Control, Microscopy

    6) Product Images from "Regulation of c-Myc Protein Abundance by a Protein Phosphatase 2A-Glycogen Synthase Kinase 3?-Negative Feedback Pathway"

    Article Title: Regulation of c-Myc Protein Abundance by a Protein Phosphatase 2A-Glycogen Synthase Kinase 3?-Negative Feedback Pathway

    Journal: Genes & Cancer

    doi: 10.1177/1947601912448067

    B56δ regulates GSK3β activity and c-Myc degradation. ( A ) 293T cells stably expressing nonsilencing shRNA (control), or B56δ shRNA (B56δ stable knockdown), and control 293T cells transfected with V5-B56δ, as indicated,
    Figure Legend Snippet: B56δ regulates GSK3β activity and c-Myc degradation. ( A ) 293T cells stably expressing nonsilencing shRNA (control), or B56δ shRNA (B56δ stable knockdown), and control 293T cells transfected with V5-B56δ, as indicated,

    Techniques Used: Activity Assay, Stable Transfection, Expressing, shRNA, Transfection

    B56δ promotes GSK3β dephosphorylation and c-Myc degradation. ( A, B ) Subconfluent 293T cells were transfected with increasing amounts of plasmid-expressing V5-His-tagged B56δ (pcDNA3.1/V5-His-B56δ) as indicated. Twenty-four
    Figure Legend Snippet: B56δ promotes GSK3β dephosphorylation and c-Myc degradation. ( A, B ) Subconfluent 293T cells were transfected with increasing amounts of plasmid-expressing V5-His-tagged B56δ (pcDNA3.1/V5-His-B56δ) as indicated. Twenty-four

    Techniques Used: De-Phosphorylation Assay, Transfection, Plasmid Preparation, Expressing

    7) Product Images from "Characterisation of a cell-free synthesised G-protein coupled receptor"

    Article Title: Characterisation of a cell-free synthesised G-protein coupled receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01227-z

    Streptavidin and His-tag pull down assay of CFPS malE-en2NTS 1 and E. coli expressed en2NTS 1 . Binding assay, testing capability of binding to Biotin-NT using Streptavidin dynabeads in combination with ( A ). CFPS produced malE-en2NTS 1 and ( B ). The E. coli expressed en2NTS 1 control. Each graph reads 1. Receptor alone. 2. Biotin-NT plus receptor. 3. Biotin NT, receptor and unlabelled NT 8–13 competitor. Binding assay using His-tag isolation dynabeads in combination with ( C ). CFPS produced malE-en2NTS 1 ( D ). E. coli expressed en2NTS 1 . Each graph reads 1. Receptor alone. 2. A647-NT 8–13 plus receptor. 3. A647-NT 8–13 , receptor and NT 8–13 competitor. Data is presented as triplicate experiments ± SEM.
    Figure Legend Snippet: Streptavidin and His-tag pull down assay of CFPS malE-en2NTS 1 and E. coli expressed en2NTS 1 . Binding assay, testing capability of binding to Biotin-NT using Streptavidin dynabeads in combination with ( A ). CFPS produced malE-en2NTS 1 and ( B ). The E. coli expressed en2NTS 1 control. Each graph reads 1. Receptor alone. 2. Biotin-NT plus receptor. 3. Biotin NT, receptor and unlabelled NT 8–13 competitor. Binding assay using His-tag isolation dynabeads in combination with ( C ). CFPS produced malE-en2NTS 1 ( D ). E. coli expressed en2NTS 1 . Each graph reads 1. Receptor alone. 2. A647-NT 8–13 plus receptor. 3. A647-NT 8–13 , receptor and NT 8–13 competitor. Data is presented as triplicate experiments ± SEM.

    Techniques Used: Pull Down Assay, Binding Assay, Produced, Isolation

    8) Product Images from "Characterisation of a cell-free synthesised G-protein coupled receptor"

    Article Title: Characterisation of a cell-free synthesised G-protein coupled receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01227-z

    Streptavidin and His-tag pull down assay of CFPS malE-en2NTS 1 and E. coli expressed en2NTS 1 . Binding assay, testing capability of binding to Biotin-NT using Streptavidin dynabeads in combination with ( A ). CFPS produced malE-en2NTS 1 and ( B ). The E. coli expressed en2NTS 1 control. Each graph reads 1. Receptor alone. 2. Biotin-NT plus receptor. 3. Biotin NT, receptor and unlabelled NT 8–13 competitor. Binding assay using His-tag isolation dynabeads in combination with ( C ). CFPS produced malE-en2NTS 1 ( D ). E. coli expressed en2NTS 1 . Each graph reads 1. Receptor alone. 2. A647-NT 8–13 plus receptor. 3. A647-NT 8–13 , receptor and NT 8–13 competitor. Data is presented as triplicate experiments ± SEM.
    Figure Legend Snippet: Streptavidin and His-tag pull down assay of CFPS malE-en2NTS 1 and E. coli expressed en2NTS 1 . Binding assay, testing capability of binding to Biotin-NT using Streptavidin dynabeads in combination with ( A ). CFPS produced malE-en2NTS 1 and ( B ). The E. coli expressed en2NTS 1 control. Each graph reads 1. Receptor alone. 2. Biotin-NT plus receptor. 3. Biotin NT, receptor and unlabelled NT 8–13 competitor. Binding assay using His-tag isolation dynabeads in combination with ( C ). CFPS produced malE-en2NTS 1 ( D ). E. coli expressed en2NTS 1 . Each graph reads 1. Receptor alone. 2. A647-NT 8–13 plus receptor. 3. A647-NT 8–13 , receptor and NT 8–13 competitor. Data is presented as triplicate experiments ± SEM.

    Techniques Used: Pull Down Assay, Binding Assay, Produced, Isolation

    9) Product Images from "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways"

    Article Title: A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

    Journal: bioRxiv

    doi: 10.1101/833806

    Intact protein MS spectra of Im7-6 synthesized and glycosylated by one-pot CFGpS reactions. ( a ) Plasmids encoding the Im7-6 target protein and sets of up to three GTs based on 12 successful biosynthetic pathways developed by two-pot GlycoPRIME screening were combined with appropriate sugar donors in a CFGpS reaction and incubated for 24 h at 30°C. ( b ) Deconvoluted intact protein spectra from Im7-6 synthesized and glycosylated in CFGpS reactions with and without ApNGT plasmid. ( c ) Deconvoluted intact protein spectra from Im7-6 synthesized and glycosylated in CFGpS reactions with ApNGT plasmid and indicated GT plasmids. ( d ) Deconvoluted intact protein spectra from Im7-6 synthesized and glycosylated in CFGpS reactions with ApNGT, NmLgtB, and indicated GT plasmids. All reactions contained equimolar amounts of each plasmid and a total plasmid concentration of 10 nM. All Im7-6 proteins were purified using Ni-NTA magnetic beads before intact protein analysis (see Methods ). All reactions showed intact protein mass shifts consistent with the modification of Im7-6 with the same glycans observed in our two-pot system ( Figs. 2 - 3 ), although at lower efficiency. MS spectra were acquired from full elution areas of all detected glycosylated and aglycosylated protein or peptide species and are representative of n=2 CFGpS reactions. Deconvoluted spectra collected from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. See Supplementary Fig. 3 for theoretical mass values.
    Figure Legend Snippet: Intact protein MS spectra of Im7-6 synthesized and glycosylated by one-pot CFGpS reactions. ( a ) Plasmids encoding the Im7-6 target protein and sets of up to three GTs based on 12 successful biosynthetic pathways developed by two-pot GlycoPRIME screening were combined with appropriate sugar donors in a CFGpS reaction and incubated for 24 h at 30°C. ( b ) Deconvoluted intact protein spectra from Im7-6 synthesized and glycosylated in CFGpS reactions with and without ApNGT plasmid. ( c ) Deconvoluted intact protein spectra from Im7-6 synthesized and glycosylated in CFGpS reactions with ApNGT plasmid and indicated GT plasmids. ( d ) Deconvoluted intact protein spectra from Im7-6 synthesized and glycosylated in CFGpS reactions with ApNGT, NmLgtB, and indicated GT plasmids. All reactions contained equimolar amounts of each plasmid and a total plasmid concentration of 10 nM. All Im7-6 proteins were purified using Ni-NTA magnetic beads before intact protein analysis (see Methods ). All reactions showed intact protein mass shifts consistent with the modification of Im7-6 with the same glycans observed in our two-pot system ( Figs. 2 - 3 ), although at lower efficiency. MS spectra were acquired from full elution areas of all detected glycosylated and aglycosylated protein or peptide species and are representative of n=2 CFGpS reactions. Deconvoluted spectra collected from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. See Supplementary Fig. 3 for theoretical mass values.

    Techniques Used: Synthesized, Incubation, Plasmid Preparation, Concentration Assay, Purification, Magnetic Beads, Modification

    Production of sialylated Im7-6 in the E. coli cytoplasm. ( a ) Design of cytoplasmic glycosylation system to produce sialylated glycoproteins in E. coli . Three plasmids containing NmNeuA (CMP-Sia synthesis), target protein containing ApNGT glycosylation acceptor sequence, and biosynthetic pathways discovered using GlycoPRIME (GT operon). ( b-f ) Deconvoluted intact protein spectra from Im7-6 purified from CLM24 ΔnanA E. coli strain containing CMP-Sia synthesis plasmid and Im7-6 target protein plasmid as well as no GT operon b ; GT operon containing ApNGT c ; GT operon containing ApNGT and LgtB d ; GT operon containing ApNGT, NmLgtB, and CjCST-I e ; or GT operon containing ApNGT, NmLgtB, and PdST6 f . The last GT in all glycosylation pathways is indicated. Mass shifts in intact protein spectra are consistent with established activities of each GT and the installation of N -linked Glc, lactose, 3’-sialyllactose, and 6’-sialyllactose onto Im7-6 in b, c, d, e, and f , respectively. All E. coli cultures were supplemented with 5 mM sialic acid and grown to OD600 = 0.6 at 37°C, induced with 1 mM IPTG and 0.2% arabinose, and then incubated overnight at 25°C. MS spectra were acquired from full elution areas of all detected glycosylated and aglycosylated protein species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. See Supplementary Table 3 for theoretical masses. Spectra representative of n=2 bacterial cultures.
    Figure Legend Snippet: Production of sialylated Im7-6 in the E. coli cytoplasm. ( a ) Design of cytoplasmic glycosylation system to produce sialylated glycoproteins in E. coli . Three plasmids containing NmNeuA (CMP-Sia synthesis), target protein containing ApNGT glycosylation acceptor sequence, and biosynthetic pathways discovered using GlycoPRIME (GT operon). ( b-f ) Deconvoluted intact protein spectra from Im7-6 purified from CLM24 ΔnanA E. coli strain containing CMP-Sia synthesis plasmid and Im7-6 target protein plasmid as well as no GT operon b ; GT operon containing ApNGT c ; GT operon containing ApNGT and LgtB d ; GT operon containing ApNGT, NmLgtB, and CjCST-I e ; or GT operon containing ApNGT, NmLgtB, and PdST6 f . The last GT in all glycosylation pathways is indicated. Mass shifts in intact protein spectra are consistent with established activities of each GT and the installation of N -linked Glc, lactose, 3’-sialyllactose, and 6’-sialyllactose onto Im7-6 in b, c, d, e, and f , respectively. All E. coli cultures were supplemented with 5 mM sialic acid and grown to OD600 = 0.6 at 37°C, induced with 1 mM IPTG and 0.2% arabinose, and then incubated overnight at 25°C. MS spectra were acquired from full elution areas of all detected glycosylated and aglycosylated protein species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. See Supplementary Table 3 for theoretical masses. Spectra representative of n=2 bacterial cultures.

    Techniques Used: Sequencing, Purification, Plasmid Preparation, Incubation

    In vitro synthesis and assembly of complex glycosylation pathways. (a) Protein name, species, previously characterized activity ( Supplementary Table 4 ) and optimized CFPS soluble yields ( Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields and errors indicate mean and S.D. from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 7 ). (b) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM appropriate sugar donors, and 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides ( Supplementary Fig. 5 ), and exoglycosidase digestions ( Supplementary Figs. 8 and 9 ) are consistent with modification of N -linked lactose with α1-3Gal; α1-4Gal; α1-3 Fuc; α2-6 Sia; α2-3 Sia and α2-8 Sia; β1-3 GlcNAc, and pyruvylation according to known GT activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. (d) Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four and five enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors and indicated GTs at half or one third the concentrations indicated in b for four and five enzyme pathways, respectively. Intact mass shifts and fragmentation spectra ( Supplementary Fig. 10 ) are consistent with fucosylation and sialylation of LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 4 - 6 and 10 - 12 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30°C, supplemented with an additional 2.5 mM sugar donors and incubated for 24 h at 30°C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of at least n=2 IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: In vitro synthesis and assembly of complex glycosylation pathways. (a) Protein name, species, previously characterized activity ( Supplementary Table 4 ) and optimized CFPS soluble yields ( Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields and errors indicate mean and S.D. from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 7 ). (b) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM appropriate sugar donors, and 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides ( Supplementary Fig. 5 ), and exoglycosidase digestions ( Supplementary Figs. 8 and 9 ) are consistent with modification of N -linked lactose with α1-3Gal; α1-4Gal; α1-3 Fuc; α2-6 Sia; α2-3 Sia and α2-8 Sia; β1-3 GlcNAc, and pyruvylation according to known GT activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. (d) Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four and five enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors and indicated GTs at half or one third the concentrations indicated in b for four and five enzyme pathways, respectively. Intact mass shifts and fragmentation spectra ( Supplementary Fig. 10 ) are consistent with fucosylation and sialylation of LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 4 - 6 and 10 - 12 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30°C, supplemented with an additional 2.5 mM sugar donors and incubated for 24 h at 30°C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of at least n=2 IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: In Vitro, Activity Assay, Purification, Modification, Produced, Incubation

    Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The α2-3 Neuraminidase S was able to remove the sialic acids installed by CjCST-I; PmST3,6; and the first sialic acid installed by CjCST-II, indicating that these enzymes were installed sialic acids with α2-3 linkages. Sialic acids installed by PdST6, HsSIAT1, as well as the second and third sialic acids installed by CjCST-II were resistant to digestion by α2-3 Neuraminidase S but were susceptible to cleavage by an α2-3,6,8 Neuraminidase which is consistent with the established α2-6 activity of PdST6 and HsSIAT1 and the α2,8 linkages installed by CjCST-II in subsequent sialic acid additions. See Methods section for exoglycosidase details. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures.
    Figure Legend Snippet: Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The α2-3 Neuraminidase S was able to remove the sialic acids installed by CjCST-I; PmST3,6; and the first sialic acid installed by CjCST-II, indicating that these enzymes were installed sialic acids with α2-3 linkages. Sialic acids installed by PdST6, HsSIAT1, as well as the second and third sialic acids installed by CjCST-II were resistant to digestion by α2-3 Neuraminidase S but were susceptible to cleavage by an α2-3,6,8 Neuraminidase which is consistent with the established α2-6 activity of PdST6 and HsSIAT1 and the α2,8 linkages installed by CjCST-II in subsequent sialic acid additions. See Methods section for exoglycosidase details. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures.

    Techniques Used: Sequencing, Modification, Purification, Magnetic Beads, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

    HdGlcNAcT does not modify the N -linked lactose substrate installed by ApNGT and NmLgtB. Deconvoluted intact protein MS spectra of IVG reaction product containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 1.5 µM HdGlcNAcT, and 2.5 mM of UDP-Glc, UDP-Gal, and UDP-GlcNAc. No peaks were detected that indicated the modification of Im7-6 with N -linked lactose installed by ApNGT and NmLgtB (see Supplementary Table 3 for theoretical mass values). Deconvoluted spectra representative of n=2 IVG reactions.
    Figure Legend Snippet: HdGlcNAcT does not modify the N -linked lactose substrate installed by ApNGT and NmLgtB. Deconvoluted intact protein MS spectra of IVG reaction product containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 1.5 µM HdGlcNAcT, and 2.5 mM of UDP-Glc, UDP-Gal, and UDP-GlcNAc. No peaks were detected that indicated the modification of Im7-6 with N -linked lactose installed by ApNGT and NmLgtB (see Supplementary Table 3 for theoretical mass values). Deconvoluted spectra representative of n=2 IVG reactions.

    Techniques Used: Modification

    Deconvoluted intact protein MS spectra of IVG reaction products showing no production fucosylated and sialylated species. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated enzymes, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified and analyzed by intact protein MS. Reactions contained 2.4 µM HpFutA and 2.4 µM PdST6 or 1.3 µM HpFutC and 0.65 µM CjCST-I as indicated. Deconvoluted spectra representative of n=2 IVGs. No peaks were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (the region of the spectra annotated in red line shows expected range of sialylated and fucosylated species) (see Supplementary Table 4 for theoretical mass values).
    Figure Legend Snippet: Deconvoluted intact protein MS spectra of IVG reaction products showing no production fucosylated and sialylated species. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated enzymes, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified and analyzed by intact protein MS. Reactions contained 2.4 µM HpFutA and 2.4 µM PdST6 or 1.3 µM HpFutC and 0.65 µM CjCST-I as indicated. Deconvoluted spectra representative of n=2 IVGs. No peaks were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (the region of the spectra annotated in red line shows expected range of sialylated and fucosylated species) (see Supplementary Table 4 for theoretical mass values).

    Techniques Used: Purification, Modification

    CjCST-I and HsSIAT1 exhibit greater activity when produced in oxidizing conditions. Deconvoluted intact protein MS spectra representative of of n=2 IVG reaction products containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM of UDP-Glc, UDP-Gal, and CMP-Sia as well as CjCST-I or HsSIAT1 made in CFPS conducted under oxidizing conditions, reducing conditions with supplemented the E. coli disulfide bond isomerase (DsbC), or standard reducing conditions (see Methods ). CFPS conditions are known to create a protein synthesis environment conducive to disulfide bond formation as previously described 24 . Lysates enriched with sialyltranferases by CFPS were added in equal volumes. Therefore, reducing reaction conditions contained 1.9 µM of CjCST-I or 3.8 µM of HsSIAT1 while oxidizing reaction conditions reactions contained 1.3 µM of CjCST-I and 0.7 µM of HsSIAT1 (detailed CFPS yield information shown in Supplementary Fig. 2 ). Aside from CFPS synthesis conditions for the CjCST-I and HsSIAT1, IVG reactions were performed identically without ensuring an oxidizing environment for glycosylation. Im7-6, ApNGT, and NmLgtB were produced with standard CFPS reaction conditions. Relative glycosylation efficiencies indicate that the oxidizing CFPS environment of CFPS allows for greater enzyme activities per unit of CFPS reaction volume and per µM of enzyme. This observation makes sense for HsSIAT1 which is normally active in the oxidizing environment of the human golgi and is known to contain disulfide bonds. Interestingly, an oxidizing synthesis environment also seems to benefit the activity of CjCST-I which does not contain disulfide bonds. However, the increased activity of CjCST-I cannot be explained by the general chaperone activity of DsbC.
    Figure Legend Snippet: CjCST-I and HsSIAT1 exhibit greater activity when produced in oxidizing conditions. Deconvoluted intact protein MS spectra representative of of n=2 IVG reaction products containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM of UDP-Glc, UDP-Gal, and CMP-Sia as well as CjCST-I or HsSIAT1 made in CFPS conducted under oxidizing conditions, reducing conditions with supplemented the E. coli disulfide bond isomerase (DsbC), or standard reducing conditions (see Methods ). CFPS conditions are known to create a protein synthesis environment conducive to disulfide bond formation as previously described 24 . Lysates enriched with sialyltranferases by CFPS were added in equal volumes. Therefore, reducing reaction conditions contained 1.9 µM of CjCST-I or 3.8 µM of HsSIAT1 while oxidizing reaction conditions reactions contained 1.3 µM of CjCST-I and 0.7 µM of HsSIAT1 (detailed CFPS yield information shown in Supplementary Fig. 2 ). Aside from CFPS synthesis conditions for the CjCST-I and HsSIAT1, IVG reactions were performed identically without ensuring an oxidizing environment for glycosylation. Im7-6, ApNGT, and NmLgtB were produced with standard CFPS reaction conditions. Relative glycosylation efficiencies indicate that the oxidizing CFPS environment of CFPS allows for greater enzyme activities per unit of CFPS reaction volume and per µM of enzyme. This observation makes sense for HsSIAT1 which is normally active in the oxidizing environment of the human golgi and is known to contain disulfide bonds. Interestingly, an oxidizing synthesis environment also seems to benefit the activity of CjCST-I which does not contain disulfide bonds. However, the increased activity of CjCST-I cannot be explained by the general chaperone activity of DsbC.

    Techniques Used: Activity Assay, Produced

    Glycopeptide MS/MS spectra of GlycoPRIME reaction products from three enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing three enzyme pathways modifying Im7-6 shown in Fig. 3 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3 and Supplementary Fig. 4 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra are representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ) and exoglycosidase sequencing ( Supplementary Figs. 8 and 9 ). All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated GTs, and appropriate sugar donors according to established GT activities.
    Figure Legend Snippet: Glycopeptide MS/MS spectra of GlycoPRIME reaction products from three enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing three enzyme pathways modifying Im7-6 shown in Fig. 3 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3 and Supplementary Fig. 4 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra are representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ) and exoglycosidase sequencing ( Supplementary Figs. 8 and 9 ). All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated GTs, and appropriate sugar donors according to established GT activities.

    Techniques Used: Tandem Mass Spectroscopy, Purification, Derivative Assay, Modification, Sequencing

    Optimization of sialyltranferase homologs. Deconvoluted intact protein MS spectra representative of n=2 IVG reactions containing 0.4 µM ApNGT, 2 µM NmLgtB, each sialyltranferase shown in Fig. 3 , and 2.5 mM each of UDP-Glc, UDP-Gal, and CMP-Sia. Lysates enriched with sialyltransferases by CFPS were added with equal volumes to each IVG reaction such that each 32 µl-IVG reaction contained a total of 25 µl of CFPS lysates. These reactions contained 12.9 µM PpST3; 9.8 µM VsST3; 1.8 µM PmST3,6; 1.3 µM CjCST-I; 5.6 µM PlST6; 0.7 µM of HsSIAT1; and 4.9 µM PdST6, based on CFPS yields shown in Supplementary Table 2 . CjCST-I and HsSIAT1 were synthesized in CFPS with oxidizing conditions because they were found to be more active when produced in this way ( Supplementary Fig. 7 ). Under the conditions above, the reaction containing PdST6 provided the most efficient conversion to 6’-siallylactose and the reaction containing CjCST-I provided the most efficient conversion to 3’-siallylactose (exoglycosidase digestions to confirm linkages are shown in Supplementary Fig. 8 ). Although only traces amounts appear in PpST6 and VsST3, MS/MS detection and identification shows that these enzymes are functional ( Supplementary Fig. 5 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: Optimization of sialyltranferase homologs. Deconvoluted intact protein MS spectra representative of n=2 IVG reactions containing 0.4 µM ApNGT, 2 µM NmLgtB, each sialyltranferase shown in Fig. 3 , and 2.5 mM each of UDP-Glc, UDP-Gal, and CMP-Sia. Lysates enriched with sialyltransferases by CFPS were added with equal volumes to each IVG reaction such that each 32 µl-IVG reaction contained a total of 25 µl of CFPS lysates. These reactions contained 12.9 µM PpST3; 9.8 µM VsST3; 1.8 µM PmST3,6; 1.3 µM CjCST-I; 5.6 µM PlST6; 0.7 µM of HsSIAT1; and 4.9 µM PdST6, based on CFPS yields shown in Supplementary Table 2 . CjCST-I and HsSIAT1 were synthesized in CFPS with oxidizing conditions because they were found to be more active when produced in this way ( Supplementary Fig. 7 ). Under the conditions above, the reaction containing PdST6 provided the most efficient conversion to 6’-siallylactose and the reaction containing CjCST-I provided the most efficient conversion to 3’-siallylactose (exoglycosidase digestions to confirm linkages are shown in Supplementary Fig. 8 ). Although only traces amounts appear in PpST6 and VsST3, MS/MS detection and identification shows that these enzymes are functional ( Supplementary Fig. 5 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: Synthesized, Produced, Tandem Mass Spectroscopy, Functional Assay

    Optimization of LgtB homolog and concentration. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and indicated concentrations of NmLgtB or NgLgtB were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NmLgtB. (b) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NgLgtB. Results representative of n=2 IVG reactions conducted for 24 h at 30°C indicate that NmLgtB produced in CFPS has greater specific activity and that nearly homogeneous N -linked lactose can be obtained with 2 µM NmLgtB. Theoretical mass values shown in Supplementary Table 3 . All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: Optimization of LgtB homolog and concentration. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and indicated concentrations of NmLgtB or NgLgtB were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NmLgtB. (b) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NgLgtB. Results representative of n=2 IVG reactions conducted for 24 h at 30°C indicate that NmLgtB produced in CFPS has greater specific activity and that nearly homogeneous N -linked lactose can be obtained with 2 µM NmLgtB. Theoretical mass values shown in Supplementary Table 3 . All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: Concentration Assay, Purification, Produced, Activity Assay

    In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. (a) Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields and errors indicate mean and standard deviation (S.D.) from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 . (b) Monosaccharide symbol key and in vitro glycosylation (IVG) reaction scheme for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. All glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. All mentions of sialic acid refer to N -acetylneuraminic acid. (c) Deconvoluted mass spectrometry spectra from Im7-6 protein purified from in vitro glycosylation (IVG) reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 UDP-Glc. Full conversion to N -linked glucose was observed after IVG incubation for 24 h at 30°C. (d) Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions assembled from CFPS reaction products with 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30°C. Observed mass shifts and MS/MS fragmentation spectra ( Supplementary Fig. 1 ) are consistent with efficient modification of N -linked glucose with β1-4Gal; β1-4Gal; β1-3GalNAc; and α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Spectra from Hpβ4GalT, Btβ4GalT1, and SpWchJ+K, which did not modify the N -linked glucose installed by ApNGT are shown in Supplementary Fig. 2 . All IVG reactions contained 10 µM Im7 and were incubated for 20 h with 2.5 mM of each appropriate nucleotide-activated sugar donor as indicated above. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n=3 independent IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. (a) Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields and errors indicate mean and standard deviation (S.D.) from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 . (b) Monosaccharide symbol key and in vitro glycosylation (IVG) reaction scheme for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. All glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. All mentions of sialic acid refer to N -acetylneuraminic acid. (c) Deconvoluted mass spectrometry spectra from Im7-6 protein purified from in vitro glycosylation (IVG) reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 UDP-Glc. Full conversion to N -linked glucose was observed after IVG incubation for 24 h at 30°C. (d) Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions assembled from CFPS reaction products with 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30°C. Observed mass shifts and MS/MS fragmentation spectra ( Supplementary Fig. 1 ) are consistent with efficient modification of N -linked glucose with β1-4Gal; β1-4Gal; β1-3GalNAc; and α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Spectra from Hpβ4GalT, Btβ4GalT1, and SpWchJ+K, which did not modify the N -linked glucose installed by ApNGT are shown in Supplementary Fig. 2 . All IVG reactions contained 10 µM Im7 and were incubated for 20 h with 2.5 mM of each appropriate nucleotide-activated sugar donor as indicated above. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n=3 independent IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: In Vitro, Activity Assay, Standard Deviation, Expressing, Mass Spectrometry, Purification, Incubation, Tandem Mass Spectroscopy, Modification

    Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways not containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The sugars installed by NmLgtB, BtGGTA, HpFutA, and HpFutC were susceptible to cleavage by commercially available β1-4 Galactosidase S; α1-3,6 Galactosidase; α1-3,4 Fucosidase; and α1-2 Fucosidase, respectfully. The galactose installed by NmLgtC was resistant to cleavage by β1-4 Galactosidase S and α1-3,6 Galactosidase, but susceptible to cleavage by α1-3,4,6 Galactosidase. The LacNAc polymer installed by alternating activities by NmLgtB and NgLgtA was susceptible to cleavage by a mixture of β1-4 Galactosidase S and the β- N -Acetylglucosaminidase S. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures. Cleavage observations are consistent with previously established GT activities ( Figs. 2 - 3 and Supplementary Table 4 ). See Methods section for exoglycosidase details.
    Figure Legend Snippet: Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways not containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The sugars installed by NmLgtB, BtGGTA, HpFutA, and HpFutC were susceptible to cleavage by commercially available β1-4 Galactosidase S; α1-3,6 Galactosidase; α1-3,4 Fucosidase; and α1-2 Fucosidase, respectfully. The galactose installed by NmLgtC was resistant to cleavage by β1-4 Galactosidase S and α1-3,6 Galactosidase, but susceptible to cleavage by α1-3,4,6 Galactosidase. The LacNAc polymer installed by alternating activities by NmLgtB and NgLgtA was susceptible to cleavage by a mixture of β1-4 Galactosidase S and the β- N -Acetylglucosaminidase S. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures. Cleavage observations are consistent with previously established GT activities ( Figs. 2 - 3 and Supplementary Table 4 ). See Methods section for exoglycosidase details.

    Techniques Used: Sequencing, Modification, Purification, Magnetic Beads, Incubation, Liquid Chromatography with Mass Spectroscopy

    Glycopeptide MS/MS spectra of GlycoPRIME reaction products from four and five enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing four and five enzyme pathways modifying Im7-6 shown in Fig. 3d and Supplementary Fig. 12 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3d and Supplementary Fig. 12 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ). Although products from five-enzyme biosynthetic pathway product could not be unambiguous defined, sugar and glycopeptide fragments do suggest modification with both fucose and sialic acids. All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated enzymes, and appropriate sugar donors according to established GT activities.
    Figure Legend Snippet: Glycopeptide MS/MS spectra of GlycoPRIME reaction products from four and five enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing four and five enzyme pathways modifying Im7-6 shown in Fig. 3d and Supplementary Fig. 12 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3d and Supplementary Fig. 12 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ). Although products from five-enzyme biosynthetic pathway product could not be unambiguous defined, sugar and glycopeptide fragments do suggest modification with both fucose and sialic acids. All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated enzymes, and appropriate sugar donors according to established GT activities.

    Techniques Used: Tandem Mass Spectroscopy, Purification, Derivative Assay, Modification

    Glycopeptide MS/MS spectra of GlycoPRIME reaction products from two enzyme biosynthetic pathways elaborating N -linked glucose. Products from IVG reactions containing two enzyme pathways modifying Im7-6 shown in Fig. 2 were purified, trypsinized, and analyzed by pseudo Multiple Reaction Monitoring (MRM) MS/MS fragmentation at theoretical glycopeptide masses (red diamonds) corresponding to detected protein MS peaks using a collisional energy of 30 eV (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. (a) MS/MS spectra of 999.49 ± 2 m/z corresponding to N -linked Glcβ1-3GalNAc installed by BfGalNAcT. (b) MS/MS spectra of 1418.29 ± 2 m/z corresponding to N -linked dextran polymer installed by Apα1-6. (c) MS/MS spectra of 985.81 ± 2 m/z corresponding with N -linked lactose installed by NmLgtB. All IVG reactions contained Im7-6, ApNGT, and appropriate sugar donors according to established enzyme activities ( Supplementary Table 4 ).
    Figure Legend Snippet: Glycopeptide MS/MS spectra of GlycoPRIME reaction products from two enzyme biosynthetic pathways elaborating N -linked glucose. Products from IVG reactions containing two enzyme pathways modifying Im7-6 shown in Fig. 2 were purified, trypsinized, and analyzed by pseudo Multiple Reaction Monitoring (MRM) MS/MS fragmentation at theoretical glycopeptide masses (red diamonds) corresponding to detected protein MS peaks using a collisional energy of 30 eV (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. (a) MS/MS spectra of 999.49 ± 2 m/z corresponding to N -linked Glcβ1-3GalNAc installed by BfGalNAcT. (b) MS/MS spectra of 1418.29 ± 2 m/z corresponding to N -linked dextran polymer installed by Apα1-6. (c) MS/MS spectra of 985.81 ± 2 m/z corresponding with N -linked lactose installed by NmLgtB. All IVG reactions contained Im7-6, ApNGT, and appropriate sugar donors according to established enzyme activities ( Supplementary Table 4 ).

    Techniques Used: Tandem Mass Spectroscopy, Purification, Derivative Assay, Modification

    GlycoPRIME screening of biosynthetic pathways containing five enzymes. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated GTs, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified from and analyzed by intact protein MS. Deconvoluted spectra representative of n=2 IVGs. (a) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 1.63 µM PdST6. (b) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 1.63 µM PdST6 (also shown in Fig. 3d ) (c) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. (d) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. Spectra in a and b as well as fragmentation spectra in Supplementary Fig. 10 indicated three and one species, respectively, which contained both sialic acid and fucose. Predicted glycosylation structures based on previously established GT activities ( Supplementary Table 4 ) and fragmentation spectra ( Supplementary Fig. 10 ). Although structures cannot be unambiguously identified, the previously observed incompatibility of HpFutA and PdST6 as well as the presence of a 1083 m/z peak (Glcβ4Galα6Sia) and the absence of a 1034 m/z (Glc(α3Fuc)β4Gal) peak in fragmentation spectra suggests that in b the proximal galactose is modified with a sialic acid while the GlcNAc is modified with the fucose. No peaks in c or d were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (see Supplementary Table 3 for theoretical mass values).
    Figure Legend Snippet: GlycoPRIME screening of biosynthetic pathways containing five enzymes. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated GTs, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified from and analyzed by intact protein MS. Deconvoluted spectra representative of n=2 IVGs. (a) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 1.63 µM PdST6. (b) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 1.63 µM PdST6 (also shown in Fig. 3d ) (c) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. (d) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. Spectra in a and b as well as fragmentation spectra in Supplementary Fig. 10 indicated three and one species, respectively, which contained both sialic acid and fucose. Predicted glycosylation structures based on previously established GT activities ( Supplementary Table 4 ) and fragmentation spectra ( Supplementary Fig. 10 ). Although structures cannot be unambiguously identified, the previously observed incompatibility of HpFutA and PdST6 as well as the presence of a 1083 m/z peak (Glcβ4Galα6Sia) and the absence of a 1034 m/z (Glc(α3Fuc)β4Gal) peak in fragmentation spectra suggests that in b the proximal galactose is modified with a sialic acid while the GlcNAc is modified with the fucose. No peaks in c or d were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (see Supplementary Table 3 for theoretical mass values).

    Techniques Used: Purification, Modification

    Deconvoluted intact protein MS spectra of IVG reaction products showing no modification of N -linked glucose installed by ApNGT. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and one elaborating GT were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra of IVG containing 1.3 µM of Hpβ4GalT. (b) Deconvoluted intact protein MS spectra of IVG containing 1.4 µM of Btβ4GalT1 supplemented with 10 µM α-lactalbumin and performed under oxidizing conditions (see Methods ). (c) Deconvoluted intact protein MS spectra of IVG containing 1.5 µM of SpWchJ and 1.0 µM of SpWchK. No peaks were detected that indicated the modification of Im7-6 with N -linked glucose installed by ApNGT (theoretical mass values shown in Supplementary Table 3 ). Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. Deconvoluted spectra shown here are representative of n=2 IVG reactions.
    Figure Legend Snippet: Deconvoluted intact protein MS spectra of IVG reaction products showing no modification of N -linked glucose installed by ApNGT. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and one elaborating GT were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra of IVG containing 1.3 µM of Hpβ4GalT. (b) Deconvoluted intact protein MS spectra of IVG containing 1.4 µM of Btβ4GalT1 supplemented with 10 µM α-lactalbumin and performed under oxidizing conditions (see Methods ). (c) Deconvoluted intact protein MS spectra of IVG containing 1.5 µM of SpWchJ and 1.0 µM of SpWchK. No peaks were detected that indicated the modification of Im7-6 with N -linked glucose installed by ApNGT (theoretical mass values shown in Supplementary Table 3 ). Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. Deconvoluted spectra shown here are representative of n=2 IVG reactions.

    Techniques Used: Modification, Purification

    10) Product Images from "Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2"

    Article Title: Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16966-3

    HULC interacts with the glycolytic enzyme PKM2. a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads ® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P
    Figure Legend Snippet: HULC interacts with the glycolytic enzyme PKM2. a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads ® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P

    Techniques Used: Synthesized, In Vitro, Incubation, Isolation, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Immunoprecipitation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Binding Assay, Magnetic Beads

    HULC interacts with the glycolytic enzyme LDHA. a Validation of the interaction between LDHA and HULC. Immunoblots of LDHA in the cell lysates and immunoprecipitates of LDHA are shown in the left panel. Agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the right panel. LincRNA-p21 was examined as the RNA control. b The cellular localizations of HULC and LDHA were analyzed by combining RNA-FISH and immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Biotinylated HULC was incubated with HepG2 cell lysate and then isolated by streptavidin-conjugated beads. LDHA and LDHB in the cell lysate and RNA pull-down were examined by western blotting. Biotinylated antisense HULC was used as the control. d His-tagged rLDHA or rLDHB was incubated with Dynabeads® His-tag isolation magnetic beads, respectively. Next, in vitro transcribed HULC or antisense HULC was incubated with the beads. Then, the RNA-protein complexes were isolated, and the levels of HULC were examined using qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P
    Figure Legend Snippet: HULC interacts with the glycolytic enzyme LDHA. a Validation of the interaction between LDHA and HULC. Immunoblots of LDHA in the cell lysates and immunoprecipitates of LDHA are shown in the left panel. Agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the right panel. LincRNA-p21 was examined as the RNA control. b The cellular localizations of HULC and LDHA were analyzed by combining RNA-FISH and immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Biotinylated HULC was incubated with HepG2 cell lysate and then isolated by streptavidin-conjugated beads. LDHA and LDHB in the cell lysate and RNA pull-down were examined by western blotting. Biotinylated antisense HULC was used as the control. d His-tagged rLDHA or rLDHB was incubated with Dynabeads® His-tag isolation magnetic beads, respectively. Next, in vitro transcribed HULC or antisense HULC was incubated with the beads. Then, the RNA-protein complexes were isolated, and the levels of HULC were examined using qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P

    Techniques Used: Western Blot, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Incubation, Isolation, Magnetic Beads, In Vitro

    11) Product Images from "Interaction of p190RhoGAP with C-terminal Domain of p120-catenin Modulates Endothelial Cytoskeleton and Permeability *"

    Article Title: Interaction of p190RhoGAP with C-terminal Domain of p120-catenin Modulates Endothelial Cytoskeleton and Permeability *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.432757

    Binding of p190RhoGAP to GST-tagged p120 catenin deletion mutants. A , GST-tagged full-length p120 catenin 1A (GST-p120 1A) and related deletion mutants were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. B , interacting complexes were pulled down with His-tagged Dynabeads, and p120 catenin domains bound to p190RhoGAP were detected by Western blot with anti-GST and anti-p120-catenin antibodies. The bottom panel shows the protein content of recombinant p120-catenin and its deletion mutants in total whole cell lysates ( WCL ). Asterisks indicate the bands corresponding to p120–1A-catenin constructs expressed. MW , molecular weight.
    Figure Legend Snippet: Binding of p190RhoGAP to GST-tagged p120 catenin deletion mutants. A , GST-tagged full-length p120 catenin 1A (GST-p120 1A) and related deletion mutants were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. B , interacting complexes were pulled down with His-tagged Dynabeads, and p120 catenin domains bound to p190RhoGAP were detected by Western blot with anti-GST and anti-p120-catenin antibodies. The bottom panel shows the protein content of recombinant p120-catenin and its deletion mutants in total whole cell lysates ( WCL ). Asterisks indicate the bands corresponding to p120–1A-catenin constructs expressed. MW , molecular weight.

    Techniques Used: Binding Assay, Western Blot, Recombinant, Construct, Molecular Weight

    Identification of the C-terminal polypeptide sequence in p120-catenin responsible for recruitment of p190RhoGAP. GST-tagged full-length p120-catenin 1A (GST-p120 1A), related deletion mutants as well as truncated mutants of p120-catenin created by site specific mutagenesis ( A ) were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. Interacting complexes were pulled down with His-tagged Dynabeads ( B ), and their content was analyzed by Western blotting using antibodies to the His tag, GST tag, p120-catenin, or p190RhoGAP as indicated. Asterisks in B indicate the bands corresponding to p120-catenin constructs expressed. The bottom panel depicts the protein content of recombinant GST-tagged p120-catenin and its deletion mutants in whole cell lysates ( WCL ).
    Figure Legend Snippet: Identification of the C-terminal polypeptide sequence in p120-catenin responsible for recruitment of p190RhoGAP. GST-tagged full-length p120-catenin 1A (GST-p120 1A), related deletion mutants as well as truncated mutants of p120-catenin created by site specific mutagenesis ( A ) were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. Interacting complexes were pulled down with His-tagged Dynabeads ( B ), and their content was analyzed by Western blotting using antibodies to the His tag, GST tag, p120-catenin, or p190RhoGAP as indicated. Asterisks in B indicate the bands corresponding to p120-catenin constructs expressed. The bottom panel depicts the protein content of recombinant GST-tagged p120-catenin and its deletion mutants in whole cell lysates ( WCL ).

    Techniques Used: Sequencing, Mutagenesis, Western Blot, Construct, Recombinant

    12) Product Images from "The function of cux1 in oxidative dna damage repair is needed to prevent premature senescence of mouse embryo fibroblasts"

    Article Title: The function of cux1 in oxidative dna damage repair is needed to prevent premature senescence of mouse embryo fibroblasts

    Journal: Oncotarget

    doi:

    Interaction between OGG1 and CUX1 (A and B) 293T cells were transfected with CUX1 (CR2CR3HD; map in Figure 2A ), FLAG-OGG1 or the empty vector, as indicated. (A) Total protein extracts were loaded on gel (input) or were submitted to immunoprecipitation with the indicated antibodies (Preimmune; IgG or CUX1) and then immunoblotted with FLAG and CUX1 antibodies. (B) Total protein extracts were submitted to immunoprecipitation with Flag antibodies in the absence or presence of ethidium bromide. (C) A pull-down assay was performed using purified GST-OGG1 and either beads bound to his-tagged CUX1-CR1CR2, HOXB3 or vector alone, followed by immunoblotting with anti-OGG1 in the presence and absence of ethidium bromide or after treatment of protein samples with benzonase.
    Figure Legend Snippet: Interaction between OGG1 and CUX1 (A and B) 293T cells were transfected with CUX1 (CR2CR3HD; map in Figure 2A ), FLAG-OGG1 or the empty vector, as indicated. (A) Total protein extracts were loaded on gel (input) or were submitted to immunoprecipitation with the indicated antibodies (Preimmune; IgG or CUX1) and then immunoblotted with FLAG and CUX1 antibodies. (B) Total protein extracts were submitted to immunoprecipitation with Flag antibodies in the absence or presence of ethidium bromide. (C) A pull-down assay was performed using purified GST-OGG1 and either beads bound to his-tagged CUX1-CR1CR2, HOXB3 or vector alone, followed by immunoblotting with anti-OGG1 in the presence and absence of ethidium bromide or after treatment of protein samples with benzonase.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Pull Down Assay, Purification

    13) Product Images from "Disrupting malaria parasite AMA1 - RON2 interaction with a small molecule prevents erythrocyte invasion"

    Article Title: Disrupting malaria parasite AMA1 - RON2 interaction with a small molecule prevents erythrocyte invasion

    Journal: Nature communications

    doi: 10.1038/ncomms3261

    Small molecules block AMA1-RON complex formation and inhibit merozoite invasion (a) Purified merozoites were used to test the effect of the three compounds on invasion of RBCs at 25 μM (white bars) and 50 μM (black bars) for 4 hr. Error bars show ± SEM from five experiments for NCGC00015280, NCGC00181034 and two for NCGC00014044. (b) Immunoprecipitation assay testing the ability of the inhibitors to block parasite AMA1-RON complex formation. Each inhibitor was used at 100 μM concentration and was immunoprecipitated using anti-RON4 antibody. RON2L peptide was used as a positive control. DMSO (1%), the solvent for the inhibitors, was used as a negative control. Experiments were performed twice and a representative western blot data is shown. (c) NCGC00015280 inhibits merozoite invasion of genetically distinct parasite clones. Purified schizonts from four different parasite clones were allowed to rupture and invade new RBCs for 4 to 6 hr in the presence of varying concentrations of the inhibitor. The number of newly invaded rings was measured by flow cytometry of SYBR green labeled parasites. IC 50 : 12 μM (FVO), 14 μM (3D7), 13 μM (DD2) and 10 μM (HB3). Error bars show ± SEM from two experiments for each parasite clone. (d) Merozoite release from schizont-infected RBCs is not affected. The effect of the inhibitors on merozoite release was tested at 30 μM, the IC 50 for invasion. Error bars represent ± SEM from three experiments for NCGC00015280, NCGC00181034 and two for NCGC00014044. The number of parasites in the absence of inhibitor was considered 100%.
    Figure Legend Snippet: Small molecules block AMA1-RON complex formation and inhibit merozoite invasion (a) Purified merozoites were used to test the effect of the three compounds on invasion of RBCs at 25 μM (white bars) and 50 μM (black bars) for 4 hr. Error bars show ± SEM from five experiments for NCGC00015280, NCGC00181034 and two for NCGC00014044. (b) Immunoprecipitation assay testing the ability of the inhibitors to block parasite AMA1-RON complex formation. Each inhibitor was used at 100 μM concentration and was immunoprecipitated using anti-RON4 antibody. RON2L peptide was used as a positive control. DMSO (1%), the solvent for the inhibitors, was used as a negative control. Experiments were performed twice and a representative western blot data is shown. (c) NCGC00015280 inhibits merozoite invasion of genetically distinct parasite clones. Purified schizonts from four different parasite clones were allowed to rupture and invade new RBCs for 4 to 6 hr in the presence of varying concentrations of the inhibitor. The number of newly invaded rings was measured by flow cytometry of SYBR green labeled parasites. IC 50 : 12 μM (FVO), 14 μM (3D7), 13 μM (DD2) and 10 μM (HB3). Error bars show ± SEM from two experiments for each parasite clone. (d) Merozoite release from schizont-infected RBCs is not affected. The effect of the inhibitors on merozoite release was tested at 30 μM, the IC 50 for invasion. Error bars represent ± SEM from three experiments for NCGC00015280, NCGC00181034 and two for NCGC00014044. The number of parasites in the absence of inhibitor was considered 100%.

    Techniques Used: Blocking Assay, Purification, Immunoprecipitation, Concentration Assay, Positive Control, Negative Control, Western Blot, Clone Assay, Flow Cytometry, Cytometry, SYBR Green Assay, Labeling, Infection

    AMA1-RON2 inhibitor blocks junction formation (a) Transmission electron microscopy of showing the different stages RBC invasion in the presence of 2 μM cytochalasin D, namely, attachment (1), re-orientation (2), junction formation (3) and rhoptry bulb secretion (4). R: rhoptry, M: micronemes, V: vacuoles; White arrow: junction. Scale bars represent 250 nm. (b) The percentage of merozoites that are attached to RBCs in the presence (white bars) or absence (black bars) of the AMA1-RON2 inhibitor NCGC00015280/NCGC00262650. (c) The percentage of apically oriented merozoites in the presence (white bars) or absence (black bars) of the inhibitor that form a junction and RBCs with vacuoles (indicative of rhoptry bulb secretion). Numbers within each bar represent the number of merozoite-attached RBCs in each category. Data was pooled from two independent experiments without inhibitor and one each with inhibitor NCGC00015280 and NCGC00262650. Scale bars represent 250 nm. (d) AMA1 secretion from micronemes is not affected. Merozoites released from schizonts in the absence (control) or presence of inhibitors NCGC00015280 (60 μM) and NCGC00181034 (60 μM) were analyzed using polyclonal antibodies to AMA1. Scale bars represent 1 μm.
    Figure Legend Snippet: AMA1-RON2 inhibitor blocks junction formation (a) Transmission electron microscopy of showing the different stages RBC invasion in the presence of 2 μM cytochalasin D, namely, attachment (1), re-orientation (2), junction formation (3) and rhoptry bulb secretion (4). R: rhoptry, M: micronemes, V: vacuoles; White arrow: junction. Scale bars represent 250 nm. (b) The percentage of merozoites that are attached to RBCs in the presence (white bars) or absence (black bars) of the AMA1-RON2 inhibitor NCGC00015280/NCGC00262650. (c) The percentage of apically oriented merozoites in the presence (white bars) or absence (black bars) of the inhibitor that form a junction and RBCs with vacuoles (indicative of rhoptry bulb secretion). Numbers within each bar represent the number of merozoite-attached RBCs in each category. Data was pooled from two independent experiments without inhibitor and one each with inhibitor NCGC00015280 and NCGC00262650. Scale bars represent 250 nm. (d) AMA1 secretion from micronemes is not affected. Merozoites released from schizonts in the absence (control) or presence of inhibitors NCGC00015280 (60 μM) and NCGC00181034 (60 μM) were analyzed using polyclonal antibodies to AMA1. Scale bars represent 1 μm.

    Techniques Used: Transmission Assay, Electron Microscopy

    Quantitative high-throughput assay to identify inhibitors of the AMA1-RON2 interaction (a) In the AlphaScreen, streptavidin-coated donor beads captures biotin-tagged RON2L peptide and the nickel-coated acceptor beads binds to His-tagged AMA1(3D7 allele). In the absence of inhibitor, excitation of the donor beads at 680nm results in production of singlet oxygen, followed by short-distance diffusion (
    Figure Legend Snippet: Quantitative high-throughput assay to identify inhibitors of the AMA1-RON2 interaction (a) In the AlphaScreen, streptavidin-coated donor beads captures biotin-tagged RON2L peptide and the nickel-coated acceptor beads binds to His-tagged AMA1(3D7 allele). In the absence of inhibitor, excitation of the donor beads at 680nm results in production of singlet oxygen, followed by short-distance diffusion (

    Techniques Used: High Throughput Screening Assay, Amplified Luminescent Proximity Homogenous Assay, Diffusion-based Assay

    Improved efficiency of analogs in blocking merozoite invasion (a) Structure of two analogs that showed improved potency. (b) Schizont-infected RBCs were allowed to rupture and invade new red cells for 4 hr in the presence of 15 μM of the parent compound (black bar) or the two analogs (grey bars). The number of newly invaded rings was measured by flow cytometry. The number of parasites in the absence of inhibitor was considered 100%. Error bars represent ± SEM from four independent experiments for each compound. (c) Purified invasive merozoites were allowed to invade RBCs and develop for 3 to 4 hr in the presence of varying concentrations of the two analogs (green and blue lines) and the parent compound (orange line). Invasion efficiency was measured by counting the number of newly formed rings. A 3 to 5-fold lower IC 50 (6 and 9.8 μM respectively) is seen for the two analogs compared to the parent compound (IC 50 : 30 μM). orange, NCGC00015280; blue, NCGC0026250 and green, NCGC00262654. Error bars represent ± SEM from three independent experiments for each compound. (d) Merozoite invasion is not inhibited by a Src Kinase Inhibitor-1, but is blocked by AMA1-RON2 inhibitors. The concentrations of the compounds used are shown in the figure. Error bars represent ± SEM from at least two experiments for each compound. The number of parasites in the absence of inhibitor was considered 100%. (e) Dihydroartemisinin (DHA) in combination with invasion inhibitors is more efficient than by itself. Inhibitors NCGC00015280 (8 μM), NCGC00262650 (8 μM) and DHA (3 nM) alone or in combination were tested for growth inhibition. Parasite growth in the absence of any inhibitor was used as a control for no inhibition. Data represents the mean ± SEM of 3D7 and FVO parasites performed in duplicates. P values were calculated using one-way ANOVA and Bonferroni’s post test was performed to compare the effect of the combination treatment over the respective individual compounds. ** P
    Figure Legend Snippet: Improved efficiency of analogs in blocking merozoite invasion (a) Structure of two analogs that showed improved potency. (b) Schizont-infected RBCs were allowed to rupture and invade new red cells for 4 hr in the presence of 15 μM of the parent compound (black bar) or the two analogs (grey bars). The number of newly invaded rings was measured by flow cytometry. The number of parasites in the absence of inhibitor was considered 100%. Error bars represent ± SEM from four independent experiments for each compound. (c) Purified invasive merozoites were allowed to invade RBCs and develop for 3 to 4 hr in the presence of varying concentrations of the two analogs (green and blue lines) and the parent compound (orange line). Invasion efficiency was measured by counting the number of newly formed rings. A 3 to 5-fold lower IC 50 (6 and 9.8 μM respectively) is seen for the two analogs compared to the parent compound (IC 50 : 30 μM). orange, NCGC00015280; blue, NCGC0026250 and green, NCGC00262654. Error bars represent ± SEM from three independent experiments for each compound. (d) Merozoite invasion is not inhibited by a Src Kinase Inhibitor-1, but is blocked by AMA1-RON2 inhibitors. The concentrations of the compounds used are shown in the figure. Error bars represent ± SEM from at least two experiments for each compound. The number of parasites in the absence of inhibitor was considered 100%. (e) Dihydroartemisinin (DHA) in combination with invasion inhibitors is more efficient than by itself. Inhibitors NCGC00015280 (8 μM), NCGC00262650 (8 μM) and DHA (3 nM) alone or in combination were tested for growth inhibition. Parasite growth in the absence of any inhibitor was used as a control for no inhibition. Data represents the mean ± SEM of 3D7 and FVO parasites performed in duplicates. P values were calculated using one-way ANOVA and Bonferroni’s post test was performed to compare the effect of the combination treatment over the respective individual compounds. ** P

    Techniques Used: Blocking Assay, Infection, Flow Cytometry, Cytometry, Purification, Inhibition

    Mode of action of the inhibitor NCGC00262650 is mediated through binding of AMA1 The mode of inhibition of the small molecule was studied by a depletion assay using either his-tagged recombinant AMA1 or biotin-tagged RON2L peptide. The ability of AMA1 or RON2 to bind the inhibitor was assessed by performing invasion assays using inhibitor-depleted supernatants. 500 pmols of either recombinant AMA1 (both 3D7 and FVO allele) or RON2L peptide bound to magnetic beads was used to deplete 500 pmols of the inhibitor (final concentration 10μM). Error bars represent ± SEM from two experiments. (b) Immunofluorescence assay using FITC-labeled RON2L peptide. FITC-labeled RON2 peptide binds to AMA1 in the mature schizonts in the absence of inhibitors, while pre-incubation with inhibitor NCGC00015280 prevents binding of the peptide. Similar results were obtained with the analog NCGC00262650 and the inhibitor NCGC00181034 (data not shown). Scale bars represent 3 μm.
    Figure Legend Snippet: Mode of action of the inhibitor NCGC00262650 is mediated through binding of AMA1 The mode of inhibition of the small molecule was studied by a depletion assay using either his-tagged recombinant AMA1 or biotin-tagged RON2L peptide. The ability of AMA1 or RON2 to bind the inhibitor was assessed by performing invasion assays using inhibitor-depleted supernatants. 500 pmols of either recombinant AMA1 (both 3D7 and FVO allele) or RON2L peptide bound to magnetic beads was used to deplete 500 pmols of the inhibitor (final concentration 10μM). Error bars represent ± SEM from two experiments. (b) Immunofluorescence assay using FITC-labeled RON2L peptide. FITC-labeled RON2 peptide binds to AMA1 in the mature schizonts in the absence of inhibitors, while pre-incubation with inhibitor NCGC00015280 prevents binding of the peptide. Similar results were obtained with the analog NCGC00262650 and the inhibitor NCGC00181034 (data not shown). Scale bars represent 3 μm.

    Techniques Used: Binding Assay, Inhibition, Depletion Assay, Recombinant, Magnetic Beads, Concentration Assay, Immunofluorescence, Labeling, Incubation

    14) Product Images from "Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2"

    Article Title: Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16966-3

    HULC interacts with the glycolytic enzyme PKM2. a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads ® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P
    Figure Legend Snippet: HULC interacts with the glycolytic enzyme PKM2. a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads ® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P

    Techniques Used: Synthesized, In Vitro, Incubation, Isolation, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Immunoprecipitation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Binding Assay, Magnetic Beads

    HULC interacts with the glycolytic enzyme LDHA. a Validation of the interaction between LDHA and HULC. Immunoblots of LDHA in the cell lysates and immunoprecipitates of LDHA are shown in the left panel. Agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the right panel. LincRNA-p21 was examined as the RNA control. b The cellular localizations of HULC and LDHA were analyzed by combining RNA-FISH and immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Biotinylated HULC was incubated with HepG2 cell lysate and then isolated by streptavidin-conjugated beads. LDHA and LDHB in the cell lysate and RNA pull-down were examined by western blotting. Biotinylated antisense HULC was used as the control. d His-tagged rLDHA or rLDHB was incubated with Dynabeads® His-tag isolation magnetic beads, respectively. Next, in vitro transcribed HULC or antisense HULC was incubated with the beads. Then, the RNA-protein complexes were isolated, and the levels of HULC were examined using qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P
    Figure Legend Snippet: HULC interacts with the glycolytic enzyme LDHA. a Validation of the interaction between LDHA and HULC. Immunoblots of LDHA in the cell lysates and immunoprecipitates of LDHA are shown in the left panel. Agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the right panel. LincRNA-p21 was examined as the RNA control. b The cellular localizations of HULC and LDHA were analyzed by combining RNA-FISH and immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Biotinylated HULC was incubated with HepG2 cell lysate and then isolated by streptavidin-conjugated beads. LDHA and LDHB in the cell lysate and RNA pull-down were examined by western blotting. Biotinylated antisense HULC was used as the control. d His-tagged rLDHA or rLDHB was incubated with Dynabeads® His-tag isolation magnetic beads, respectively. Next, in vitro transcribed HULC or antisense HULC was incubated with the beads. Then, the RNA-protein complexes were isolated, and the levels of HULC were examined using qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P

    Techniques Used: Western Blot, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Incubation, Isolation, Magnetic Beads, In Vitro

    15) Product Images from "FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance"

    Article Title: FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv076

    Polη interacts with the POLD2 subunit of Polδ. ( A ) Polη full length or truncation mutants and POLD2 proteins were expressed in the yeast strain AH109 as a transcription activation domain fusion protein (in pACT2) and a DNA binding domain fusion protein (in pGBKT7), respectively. Yeast transformants expressing both Polη and POLD2 fusion proteins are selected on double drop out medium (-W-L). Positive interactions are indicated by growth on quadruple drop out medium (-W-L-A-H). ( B ) Minimum amino acid sequence of the Polη region that interacts with POLD2. Residues that were mutated to alanine in the full-length Polη coding sequence and tested for their interaction with POLD2 are boxed. ( C ) Wild type and FF 483–484 AA (Polη F1*) of full-length Polη were examined for the interaction with POLD2. ( D ) Association of POLD2 with Polη in vitro . Left panel: physical interaction between the purified human DNA polymerase η and the POLD2 subunit of DNA polymerase δ. GST pull-down experiment was carried out using Flag-POLD2 and GST-Polη or GST followed by immobilization on GTH beads. The bound proteins were analyzed by immunoblotting or Coomassie blue staining. Right panel: selective binding of POLD2 with His-GST-Polη 393–511 . Pull-down experiments were carried out using in vitro translated POLD2, His-GST, His-GST-Polη 393–511 or His-GST-Polη 393–511 (F1*) followed by immobilization on IMAC magnetic beads. Input and bound proteins were analyzed by Coomassie blue staining or immunoblotting. Inputs correspond to 1/10th the protein amount used for pull-down.
    Figure Legend Snippet: Polη interacts with the POLD2 subunit of Polδ. ( A ) Polη full length or truncation mutants and POLD2 proteins were expressed in the yeast strain AH109 as a transcription activation domain fusion protein (in pACT2) and a DNA binding domain fusion protein (in pGBKT7), respectively. Yeast transformants expressing both Polη and POLD2 fusion proteins are selected on double drop out medium (-W-L). Positive interactions are indicated by growth on quadruple drop out medium (-W-L-A-H). ( B ) Minimum amino acid sequence of the Polη region that interacts with POLD2. Residues that were mutated to alanine in the full-length Polη coding sequence and tested for their interaction with POLD2 are boxed. ( C ) Wild type and FF 483–484 AA (Polη F1*) of full-length Polη were examined for the interaction with POLD2. ( D ) Association of POLD2 with Polη in vitro . Left panel: physical interaction between the purified human DNA polymerase η and the POLD2 subunit of DNA polymerase δ. GST pull-down experiment was carried out using Flag-POLD2 and GST-Polη or GST followed by immobilization on GTH beads. The bound proteins were analyzed by immunoblotting or Coomassie blue staining. Right panel: selective binding of POLD2 with His-GST-Polη 393–511 . Pull-down experiments were carried out using in vitro translated POLD2, His-GST, His-GST-Polη 393–511 or His-GST-Polη 393–511 (F1*) followed by immobilization on IMAC magnetic beads. Input and bound proteins were analyzed by Coomassie blue staining or immunoblotting. Inputs correspond to 1/10th the protein amount used for pull-down.

    Techniques Used: Activation Assay, Binding Assay, Expressing, Sequencing, In Vitro, Purification, Staining, Magnetic Beads

    16) Product Images from "Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization"

    Article Title: Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0491-9

    Anti- Campylobacter nanobodies interact with native outer membrane proteins. Serial tenfold dilutions of the nanobodies were used in ELISA to assess the binding with linear or conformational epitopes. OMPs (1 µg/mL) were coated in a 96-well plate and the interaction of His-tagged nanobodies with native, untreated OMP, and with denatured protein extract was measured. Binding of A Nb5, B Nb22, C Nb23, D Nb24, E Nb49 and F Nb84 was measured. For detection, mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase were used. The error bars represent the standard deviations.
    Figure Legend Snippet: Anti- Campylobacter nanobodies interact with native outer membrane proteins. Serial tenfold dilutions of the nanobodies were used in ELISA to assess the binding with linear or conformational epitopes. OMPs (1 µg/mL) were coated in a 96-well plate and the interaction of His-tagged nanobodies with native, untreated OMP, and with denatured protein extract was measured. Binding of A Nb5, B Nb22, C Nb23, D Nb24, E Nb49 and F Nb84 was measured. For detection, mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase were used. The error bars represent the standard deviations.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.
    Figure Legend Snippet: Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.

    Techniques Used: Agglutination, Negative Control, Microscopy

    Amino acid sequence alignment of anti- Campylobacter nanobodies selected for their broad specificity. The structural framework regions are indicated by FR1–FR4 and the red boxes specify the CDRs. On the basis of the variation of the amino acid sequence of the CDR3, the nanobodies were divided in twelve unique groups.
    Figure Legend Snippet: Amino acid sequence alignment of anti- Campylobacter nanobodies selected for their broad specificity. The structural framework regions are indicated by FR1–FR4 and the red boxes specify the CDRs. On the basis of the variation of the amino acid sequence of the CDR3, the nanobodies were divided in twelve unique groups.

    Techniques Used: Sequencing

    Detection of the interaction of anti- Campylobacter nanobodies and C. jejuni KC40 by immunofluorescence microscopy. The interaction was detected by ( A , C , E , G ) immunofluorescence microscopy and the C. jejuni cells ( B , D , F , H ) were visualised by bright field microscopy. A , B A nanobody specific for F4-fimbriated enterotoxigenic E. coli shows no binding with the C. jejuni cells. C , D ; E , F and G , H The anti- Campylobacter nanobodies Nb22, Nb23 and Nb84 respectively, binds specifically with the C. jejuni cells.
    Figure Legend Snippet: Detection of the interaction of anti- Campylobacter nanobodies and C. jejuni KC40 by immunofluorescence microscopy. The interaction was detected by ( A , C , E , G ) immunofluorescence microscopy and the C. jejuni cells ( B , D , F , H ) were visualised by bright field microscopy. A , B A nanobody specific for F4-fimbriated enterotoxigenic E. coli shows no binding with the C. jejuni cells. C , D ; E , F and G , H The anti- Campylobacter nanobodies Nb22, Nb23 and Nb84 respectively, binds specifically with the C. jejuni cells.

    Techniques Used: Immunofluorescence, Microscopy, Binding Assay

    17) Product Images from "The function of cux1 in oxidative dna damage repair is needed to prevent premature senescence of mouse embryo fibroblasts"

    Article Title: The function of cux1 in oxidative dna damage repair is needed to prevent premature senescence of mouse embryo fibroblasts

    Journal: Oncotarget

    doi:

    The proliferation block of Cux1−/− MEFs in 20% oxygen is rescued by CUX1 and by the Cut repeats 1 and 2 Cux1 −/− MEFs were stably infected in 3% oxygen with a retrovirus expressing p200 CUX1, CR1CR2-NLS or an empty vector. Following selection, cells were maintained in 3% or 20% oxygen and counted over a period of 19 days, from days 30 to 49.
    Figure Legend Snippet: The proliferation block of Cux1−/− MEFs in 20% oxygen is rescued by CUX1 and by the Cut repeats 1 and 2 Cux1 −/− MEFs were stably infected in 3% oxygen with a retrovirus expressing p200 CUX1, CR1CR2-NLS or an empty vector. Following selection, cells were maintained in 3% or 20% oxygen and counted over a period of 19 days, from days 30 to 49.

    Techniques Used: Blocking Assay, Stable Transfection, Infection, Expressing, Plasmid Preparation, Selection

    Rescue of DNA repair defect by recombinant CUX1 proteins (A) Schematic representation of CUX1 proteins used in this study. Note that the proteins expressed in bacteria contain a histidine tag at their N-terminus. The CR1CR2 expressed in mammalian cells contains in addition a nuclear localization signal at its C-terminus. Shown at the top are the functional domains: Inh, auto-inhibitory domain; CC, coiled-coil; CR1, CR2 and CR3, Cut repeat 1, 2 and 3; HD, Cut homeodomain; R1 and R2, repression domains 1 and 2. Note that the auto-inhibitory domain prevents binding by covalently linked DNA binding domains [ 35 ]. (B) Cux1 −/− MEFs were cultured in 3% oxygen and stably infected with retroviruses expressing p200 CUX1-HA, p110 CUX1-HA, CR1CR2-NLS-HA or nothing (vector). Expression of recombinant CUX1 protein expression was analyzed by immunoblotting using HA antibodies. (C, D and E) Comet assays were performed after 32 days of culture in three conditions as described in Figure 1 . Comet tail moments were scored for at least 50 cells per conditions. Error bars represent standard error. *** p
    Figure Legend Snippet: Rescue of DNA repair defect by recombinant CUX1 proteins (A) Schematic representation of CUX1 proteins used in this study. Note that the proteins expressed in bacteria contain a histidine tag at their N-terminus. The CR1CR2 expressed in mammalian cells contains in addition a nuclear localization signal at its C-terminus. Shown at the top are the functional domains: Inh, auto-inhibitory domain; CC, coiled-coil; CR1, CR2 and CR3, Cut repeat 1, 2 and 3; HD, Cut homeodomain; R1 and R2, repression domains 1 and 2. Note that the auto-inhibitory domain prevents binding by covalently linked DNA binding domains [ 35 ]. (B) Cux1 −/− MEFs were cultured in 3% oxygen and stably infected with retroviruses expressing p200 CUX1-HA, p110 CUX1-HA, CR1CR2-NLS-HA or nothing (vector). Expression of recombinant CUX1 protein expression was analyzed by immunoblotting using HA antibodies. (C, D and E) Comet assays were performed after 32 days of culture in three conditions as described in Figure 1 . Comet tail moments were scored for at least 50 cells per conditions. Error bars represent standard error. *** p

    Techniques Used: Recombinant, Functional Assay, Binding Assay, Cell Culture, Stable Transfection, Infection, Expressing, Plasmid Preparation

    Interaction between OGG1 and CUX1 (A and B) 293T cells were transfected with CUX1 (CR2CR3HD; map in Figure 2A ), FLAG-OGG1 or the empty vector, as indicated. (A) Total protein extracts were loaded on gel (input) or were submitted to immunoprecipitation with the indicated antibodies (Preimmune; IgG or CUX1) and then immunoblotted with FLAG and CUX1 antibodies. (B) Total protein extracts were submitted to immunoprecipitation with Flag antibodies in the absence or presence of ethidium bromide. (C) A pull-down assay was performed using purified GST-OGG1 and either beads bound to his-tagged CUX1-CR1CR2, HOXB3 or vector alone, followed by immunoblotting with anti-OGG1 in the presence and absence of ethidium bromide or after treatment of protein samples with benzonase.
    Figure Legend Snippet: Interaction between OGG1 and CUX1 (A and B) 293T cells were transfected with CUX1 (CR2CR3HD; map in Figure 2A ), FLAG-OGG1 or the empty vector, as indicated. (A) Total protein extracts were loaded on gel (input) or were submitted to immunoprecipitation with the indicated antibodies (Preimmune; IgG or CUX1) and then immunoblotted with FLAG and CUX1 antibodies. (B) Total protein extracts were submitted to immunoprecipitation with Flag antibodies in the absence or presence of ethidium bromide. (C) A pull-down assay was performed using purified GST-OGG1 and either beads bound to his-tagged CUX1-CR1CR2, HOXB3 or vector alone, followed by immunoblotting with anti-OGG1 in the presence and absence of ethidium bromide or after treatment of protein samples with benzonase.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Pull Down Assay, Purification

    18) Product Images from "CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage *"

    Article Title: CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.651042

    Cux2 knockdown increases DNA damage in embryonic cortical neurons. Rat cortical neurons were infected with lentiviral vectors expressing Cux2 shRNA or control non-targeting sequence ( NTS ). On day 4, RNA was isolated, and cells were submitted to single
    Figure Legend Snippet: Cux2 knockdown increases DNA damage in embryonic cortical neurons. Rat cortical neurons were infected with lentiviral vectors expressing Cux2 shRNA or control non-targeting sequence ( NTS ). On day 4, RNA was isolated, and cells were submitted to single

    Techniques Used: Infection, Expressing, shRNA, Sequencing, Isolation

    Stimulation of enzymatic activity of OGG1 by CUX2 Cut repeat domains. A , schematic representation of the 8-oxoG fluorescence-based cleavage assay. The top strand contains the carboxytetramethylrhodamine ( TAMRA ) fluorescent reporter at its 5′-end
    Figure Legend Snippet: Stimulation of enzymatic activity of OGG1 by CUX2 Cut repeat domains. A , schematic representation of the 8-oxoG fluorescence-based cleavage assay. The top strand contains the carboxytetramethylrhodamine ( TAMRA ) fluorescent reporter at its 5′-end

    Techniques Used: Activity Assay, Fluorescence, Cleavage Assay

    Ectopic expression of CUX2 proteins accelerates DNA repair. HCC38 cells were stably infected with retroviruses expressing CUX2 CR1CR2-NLS-HA, CUX2 CR2CR3HD-NLS-HA, or nothing (vector). A , expression of recombinant CUX2 protein expression was analyzed
    Figure Legend Snippet: Ectopic expression of CUX2 proteins accelerates DNA repair. HCC38 cells were stably infected with retroviruses expressing CUX2 CR1CR2-NLS-HA, CUX2 CR2CR3HD-NLS-HA, or nothing (vector). A , expression of recombinant CUX2 protein expression was analyzed

    Techniques Used: Expressing, Stable Transfection, Infection, Plasmid Preparation, Recombinant

    CUX2 interacts directly with OGG1 and stimulates its binding to DNA. A , HEK293T cells were transiently transfected with an empty vector or a vector expressing CUX2-HA. Total protein extracts were subjected to immunoprecipitation ( IP ) with OGG1 antibody
    Figure Legend Snippet: CUX2 interacts directly with OGG1 and stimulates its binding to DNA. A , HEK293T cells were transiently transfected with an empty vector or a vector expressing CUX2-HA. Total protein extracts were subjected to immunoprecipitation ( IP ) with OGG1 antibody

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

    Cux2 −/− mouse embryo fibroblasts exhibit a defect in DNA repair. MEFs from Cux2 +/+ and Cux2 −/− mice were maintained in 3% oxygen, then exposed to 10 μ m H 2 O 2 for 20 min, allowed to recover for the indicated time,
    Figure Legend Snippet: Cux2 −/− mouse embryo fibroblasts exhibit a defect in DNA repair. MEFs from Cux2 +/+ and Cux2 −/− mice were maintained in 3% oxygen, then exposed to 10 μ m H 2 O 2 for 20 min, allowed to recover for the indicated time,

    Techniques Used: Mouse Assay

    CUX2 knockdown reduces proliferation and increases apoptosis in HCC38 breast tumor cells. HCC38 breast tumor cells were infected with lentiviruses expressing shCUX2 or shLuciferase ( shLUC ) RNA. A , CUX2 mRNA and protein expression were measured on day
    Figure Legend Snippet: CUX2 knockdown reduces proliferation and increases apoptosis in HCC38 breast tumor cells. HCC38 breast tumor cells were infected with lentiviruses expressing shCUX2 or shLuciferase ( shLUC ) RNA. A , CUX2 mRNA and protein expression were measured on day

    Techniques Used: Infection, Expressing

    CUX2 knockdown delays repair of oxidative DNA damage. HCC38 breast tumor cells were infected with lentiviruses expressing shCUX2 or shLuciferase ( sh Luc ) RNA. Following exposure to 50 μ m H 2 O 2 , cells were allowed to recover for the indicated time
    Figure Legend Snippet: CUX2 knockdown delays repair of oxidative DNA damage. HCC38 breast tumor cells were infected with lentiviruses expressing shCUX2 or shLuciferase ( sh Luc ) RNA. Following exposure to 50 μ m H 2 O 2 , cells were allowed to recover for the indicated time

    Techniques Used: Infection, Expressing

    Ectopic expression of CUX2 proteins rescues the DNA repair defect of Cux2 −/− MEFs. Cux2 −/− MEFs were stably infected with retroviruses expressing CUX2 CR1CR2-NLS-HA, CUX2 CR2CR3HD-NLS-HA, CUX2 CR3HD-NLS-HA, or lacZ (control).
    Figure Legend Snippet: Ectopic expression of CUX2 proteins rescues the DNA repair defect of Cux2 −/− MEFs. Cux2 −/− MEFs were stably infected with retroviruses expressing CUX2 CR1CR2-NLS-HA, CUX2 CR2CR3HD-NLS-HA, CUX2 CR3HD-NLS-HA, or lacZ (control).

    Techniques Used: Expressing, Stable Transfection, Infection

    Cut repeat domains of CUX2 stimulate the glycosylase and AP lyase activities of OGG1. A , diagrammatic representation of CUX2 proteins. Shown at the top are the evolutionarily conserved domains: coiled coil ( CC ), CR1, CR2, CR3, and HD. CR1 Mut contains
    Figure Legend Snippet: Cut repeat domains of CUX2 stimulate the glycosylase and AP lyase activities of OGG1. A , diagrammatic representation of CUX2 proteins. Shown at the top are the evolutionarily conserved domains: coiled coil ( CC ), CR1, CR2, CR3, and HD. CR1 Mut contains

    Techniques Used:

    19) Product Images from "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways"

    Article Title: A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

    Journal: bioRxiv

    doi: 10.1101/833806

    In vitro synthesis and assembly of complex glycosylation pathways. (a) Protein name, species, previously characterized activity ( Supplementary Table 4 ) and optimized CFPS soluble yields ( Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields and errors indicate mean and S.D. from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 7 ). (b) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM appropriate sugar donors, and 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides ( Supplementary Fig. 5 ), and exoglycosidase digestions ( Supplementary Figs. 8 and 9 ) are consistent with modification of N -linked lactose with α1-3Gal; α1-4Gal; α1-3 Fuc; α2-6 Sia; α2-3 Sia and α2-8 Sia; β1-3 GlcNAc, and pyruvylation according to known GT activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. (d) Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four and five enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors and indicated GTs at half or one third the concentrations indicated in b for four and five enzyme pathways, respectively. Intact mass shifts and fragmentation spectra ( Supplementary Fig. 10 ) are consistent with fucosylation and sialylation of LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 4 - 6 and 10 - 12 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30°C, supplemented with an additional 2.5 mM sugar donors and incubated for 24 h at 30°C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of at least n=2 IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: In vitro synthesis and assembly of complex glycosylation pathways. (a) Protein name, species, previously characterized activity ( Supplementary Table 4 ) and optimized CFPS soluble yields ( Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields and errors indicate mean and S.D. from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 7 ). (b) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM appropriate sugar donors, and 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides ( Supplementary Fig. 5 ), and exoglycosidase digestions ( Supplementary Figs. 8 and 9 ) are consistent with modification of N -linked lactose with α1-3Gal; α1-4Gal; α1-3 Fuc; α2-6 Sia; α2-3 Sia and α2-8 Sia; β1-3 GlcNAc, and pyruvylation according to known GT activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. (d) Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four and five enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors and indicated GTs at half or one third the concentrations indicated in b for four and five enzyme pathways, respectively. Intact mass shifts and fragmentation spectra ( Supplementary Fig. 10 ) are consistent with fucosylation and sialylation of LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 4 - 6 and 10 - 12 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30°C, supplemented with an additional 2.5 mM sugar donors and incubated for 24 h at 30°C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of at least n=2 IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: In Vitro, Activity Assay, Purification, Modification, Produced, Incubation

    Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The α2-3 Neuraminidase S was able to remove the sialic acids installed by CjCST-I; PmST3,6; and the first sialic acid installed by CjCST-II, indicating that these enzymes were installed sialic acids with α2-3 linkages. Sialic acids installed by PdST6, HsSIAT1, as well as the second and third sialic acids installed by CjCST-II were resistant to digestion by α2-3 Neuraminidase S but were susceptible to cleavage by an α2-3,6,8 Neuraminidase which is consistent with the established α2-6 activity of PdST6 and HsSIAT1 and the α2,8 linkages installed by CjCST-II in subsequent sialic acid additions. See Methods section for exoglycosidase details. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures.
    Figure Legend Snippet: Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The α2-3 Neuraminidase S was able to remove the sialic acids installed by CjCST-I; PmST3,6; and the first sialic acid installed by CjCST-II, indicating that these enzymes were installed sialic acids with α2-3 linkages. Sialic acids installed by PdST6, HsSIAT1, as well as the second and third sialic acids installed by CjCST-II were resistant to digestion by α2-3 Neuraminidase S but were susceptible to cleavage by an α2-3,6,8 Neuraminidase which is consistent with the established α2-6 activity of PdST6 and HsSIAT1 and the α2,8 linkages installed by CjCST-II in subsequent sialic acid additions. See Methods section for exoglycosidase details. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures.

    Techniques Used: Sequencing, Modification, Purification, Magnetic Beads, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

    HdGlcNAcT does not modify the N -linked lactose substrate installed by ApNGT and NmLgtB. Deconvoluted intact protein MS spectra of IVG reaction product containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 1.5 µM HdGlcNAcT, and 2.5 mM of UDP-Glc, UDP-Gal, and UDP-GlcNAc. No peaks were detected that indicated the modification of Im7-6 with N -linked lactose installed by ApNGT and NmLgtB (see Supplementary Table 3 for theoretical mass values). Deconvoluted spectra representative of n=2 IVG reactions.
    Figure Legend Snippet: HdGlcNAcT does not modify the N -linked lactose substrate installed by ApNGT and NmLgtB. Deconvoluted intact protein MS spectra of IVG reaction product containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 1.5 µM HdGlcNAcT, and 2.5 mM of UDP-Glc, UDP-Gal, and UDP-GlcNAc. No peaks were detected that indicated the modification of Im7-6 with N -linked lactose installed by ApNGT and NmLgtB (see Supplementary Table 3 for theoretical mass values). Deconvoluted spectra representative of n=2 IVG reactions.

    Techniques Used: Modification

    Deconvoluted intact protein MS spectra of IVG reaction products showing no production fucosylated and sialylated species. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated enzymes, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified and analyzed by intact protein MS. Reactions contained 2.4 µM HpFutA and 2.4 µM PdST6 or 1.3 µM HpFutC and 0.65 µM CjCST-I as indicated. Deconvoluted spectra representative of n=2 IVGs. No peaks were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (the region of the spectra annotated in red line shows expected range of sialylated and fucosylated species) (see Supplementary Table 4 for theoretical mass values).
    Figure Legend Snippet: Deconvoluted intact protein MS spectra of IVG reaction products showing no production fucosylated and sialylated species. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated enzymes, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified and analyzed by intact protein MS. Reactions contained 2.4 µM HpFutA and 2.4 µM PdST6 or 1.3 µM HpFutC and 0.65 µM CjCST-I as indicated. Deconvoluted spectra representative of n=2 IVGs. No peaks were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (the region of the spectra annotated in red line shows expected range of sialylated and fucosylated species) (see Supplementary Table 4 for theoretical mass values).

    Techniques Used: Purification, Modification

    CjCST-I and HsSIAT1 exhibit greater activity when produced in oxidizing conditions. Deconvoluted intact protein MS spectra representative of of n=2 IVG reaction products containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM of UDP-Glc, UDP-Gal, and CMP-Sia as well as CjCST-I or HsSIAT1 made in CFPS conducted under oxidizing conditions, reducing conditions with supplemented the E. coli disulfide bond isomerase (DsbC), or standard reducing conditions (see Methods ). CFPS conditions are known to create a protein synthesis environment conducive to disulfide bond formation as previously described 24 . Lysates enriched with sialyltranferases by CFPS were added in equal volumes. Therefore, reducing reaction conditions contained 1.9 µM of CjCST-I or 3.8 µM of HsSIAT1 while oxidizing reaction conditions reactions contained 1.3 µM of CjCST-I and 0.7 µM of HsSIAT1 (detailed CFPS yield information shown in Supplementary Fig. 2 ). Aside from CFPS synthesis conditions for the CjCST-I and HsSIAT1, IVG reactions were performed identically without ensuring an oxidizing environment for glycosylation. Im7-6, ApNGT, and NmLgtB were produced with standard CFPS reaction conditions. Relative glycosylation efficiencies indicate that the oxidizing CFPS environment of CFPS allows for greater enzyme activities per unit of CFPS reaction volume and per µM of enzyme. This observation makes sense for HsSIAT1 which is normally active in the oxidizing environment of the human golgi and is known to contain disulfide bonds. Interestingly, an oxidizing synthesis environment also seems to benefit the activity of CjCST-I which does not contain disulfide bonds. However, the increased activity of CjCST-I cannot be explained by the general chaperone activity of DsbC.
    Figure Legend Snippet: CjCST-I and HsSIAT1 exhibit greater activity when produced in oxidizing conditions. Deconvoluted intact protein MS spectra representative of of n=2 IVG reaction products containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, 2.5 mM of UDP-Glc, UDP-Gal, and CMP-Sia as well as CjCST-I or HsSIAT1 made in CFPS conducted under oxidizing conditions, reducing conditions with supplemented the E. coli disulfide bond isomerase (DsbC), or standard reducing conditions (see Methods ). CFPS conditions are known to create a protein synthesis environment conducive to disulfide bond formation as previously described 24 . Lysates enriched with sialyltranferases by CFPS were added in equal volumes. Therefore, reducing reaction conditions contained 1.9 µM of CjCST-I or 3.8 µM of HsSIAT1 while oxidizing reaction conditions reactions contained 1.3 µM of CjCST-I and 0.7 µM of HsSIAT1 (detailed CFPS yield information shown in Supplementary Fig. 2 ). Aside from CFPS synthesis conditions for the CjCST-I and HsSIAT1, IVG reactions were performed identically without ensuring an oxidizing environment for glycosylation. Im7-6, ApNGT, and NmLgtB were produced with standard CFPS reaction conditions. Relative glycosylation efficiencies indicate that the oxidizing CFPS environment of CFPS allows for greater enzyme activities per unit of CFPS reaction volume and per µM of enzyme. This observation makes sense for HsSIAT1 which is normally active in the oxidizing environment of the human golgi and is known to contain disulfide bonds. Interestingly, an oxidizing synthesis environment also seems to benefit the activity of CjCST-I which does not contain disulfide bonds. However, the increased activity of CjCST-I cannot be explained by the general chaperone activity of DsbC.

    Techniques Used: Activity Assay, Produced

    Glycopeptide MS/MS spectra of GlycoPRIME reaction products from three enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing three enzyme pathways modifying Im7-6 shown in Fig. 3 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3 and Supplementary Fig. 4 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra are representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ) and exoglycosidase sequencing ( Supplementary Figs. 8 and 9 ). All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated GTs, and appropriate sugar donors according to established GT activities.
    Figure Legend Snippet: Glycopeptide MS/MS spectra of GlycoPRIME reaction products from three enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing three enzyme pathways modifying Im7-6 shown in Fig. 3 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3 and Supplementary Fig. 4 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra are representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ) and exoglycosidase sequencing ( Supplementary Figs. 8 and 9 ). All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated GTs, and appropriate sugar donors according to established GT activities.

    Techniques Used: Tandem Mass Spectroscopy, Purification, Derivative Assay, Modification, Sequencing

    Optimization of sialyltranferase homologs. Deconvoluted intact protein MS spectra representative of n=2 IVG reactions containing 0.4 µM ApNGT, 2 µM NmLgtB, each sialyltranferase shown in Fig. 3 , and 2.5 mM each of UDP-Glc, UDP-Gal, and CMP-Sia. Lysates enriched with sialyltransferases by CFPS were added with equal volumes to each IVG reaction such that each 32 µl-IVG reaction contained a total of 25 µl of CFPS lysates. These reactions contained 12.9 µM PpST3; 9.8 µM VsST3; 1.8 µM PmST3,6; 1.3 µM CjCST-I; 5.6 µM PlST6; 0.7 µM of HsSIAT1; and 4.9 µM PdST6, based on CFPS yields shown in Supplementary Table 2 . CjCST-I and HsSIAT1 were synthesized in CFPS with oxidizing conditions because they were found to be more active when produced in this way ( Supplementary Fig. 7 ). Under the conditions above, the reaction containing PdST6 provided the most efficient conversion to 6’-siallylactose and the reaction containing CjCST-I provided the most efficient conversion to 3’-siallylactose (exoglycosidase digestions to confirm linkages are shown in Supplementary Fig. 8 ). Although only traces amounts appear in PpST6 and VsST3, MS/MS detection and identification shows that these enzymes are functional ( Supplementary Fig. 5 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: Optimization of sialyltranferase homologs. Deconvoluted intact protein MS spectra representative of n=2 IVG reactions containing 0.4 µM ApNGT, 2 µM NmLgtB, each sialyltranferase shown in Fig. 3 , and 2.5 mM each of UDP-Glc, UDP-Gal, and CMP-Sia. Lysates enriched with sialyltransferases by CFPS were added with equal volumes to each IVG reaction such that each 32 µl-IVG reaction contained a total of 25 µl of CFPS lysates. These reactions contained 12.9 µM PpST3; 9.8 µM VsST3; 1.8 µM PmST3,6; 1.3 µM CjCST-I; 5.6 µM PlST6; 0.7 µM of HsSIAT1; and 4.9 µM PdST6, based on CFPS yields shown in Supplementary Table 2 . CjCST-I and HsSIAT1 were synthesized in CFPS with oxidizing conditions because they were found to be more active when produced in this way ( Supplementary Fig. 7 ). Under the conditions above, the reaction containing PdST6 provided the most efficient conversion to 6’-siallylactose and the reaction containing CjCST-I provided the most efficient conversion to 3’-siallylactose (exoglycosidase digestions to confirm linkages are shown in Supplementary Fig. 8 ). Although only traces amounts appear in PpST6 and VsST3, MS/MS detection and identification shows that these enzymes are functional ( Supplementary Fig. 5 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: Synthesized, Produced, Tandem Mass Spectroscopy, Functional Assay

    Optimization of LgtB homolog and concentration. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and indicated concentrations of NmLgtB or NgLgtB were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NmLgtB. (b) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NgLgtB. Results representative of n=2 IVG reactions conducted for 24 h at 30°C indicate that NmLgtB produced in CFPS has greater specific activity and that nearly homogeneous N -linked lactose can be obtained with 2 µM NmLgtB. Theoretical mass values shown in Supplementary Table 3 . All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: Optimization of LgtB homolog and concentration. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and indicated concentrations of NmLgtB or NgLgtB were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NmLgtB. (b) Deconvoluted intact protein MS spectra from IVG reactions containing indicated concentrations of NgLgtB. Results representative of n=2 IVG reactions conducted for 24 h at 30°C indicate that NmLgtB produced in CFPS has greater specific activity and that nearly homogeneous N -linked lactose can be obtained with 2 µM NmLgtB. Theoretical mass values shown in Supplementary Table 3 . All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and were deconvoluted from m/z 100-2000 into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: Concentration Assay, Purification, Produced, Activity Assay

    In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. (a) Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields and errors indicate mean and standard deviation (S.D.) from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 . (b) Monosaccharide symbol key and in vitro glycosylation (IVG) reaction scheme for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. All glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. All mentions of sialic acid refer to N -acetylneuraminic acid. (c) Deconvoluted mass spectrometry spectra from Im7-6 protein purified from in vitro glycosylation (IVG) reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 UDP-Glc. Full conversion to N -linked glucose was observed after IVG incubation for 24 h at 30°C. (d) Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions assembled from CFPS reaction products with 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30°C. Observed mass shifts and MS/MS fragmentation spectra ( Supplementary Fig. 1 ) are consistent with efficient modification of N -linked glucose with β1-4Gal; β1-4Gal; β1-3GalNAc; and α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Spectra from Hpβ4GalT, Btβ4GalT1, and SpWchJ+K, which did not modify the N -linked glucose installed by ApNGT are shown in Supplementary Fig. 2 . All IVG reactions contained 10 µM Im7 and were incubated for 20 h with 2.5 mM of each appropriate nucleotide-activated sugar donor as indicated above. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n=3 independent IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.
    Figure Legend Snippet: In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. (a) Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields and errors indicate mean and standard deviation (S.D.) from n=3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 . (b) Monosaccharide symbol key and in vitro glycosylation (IVG) reaction scheme for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. All glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. All mentions of sialic acid refer to N -acetylneuraminic acid. (c) Deconvoluted mass spectrometry spectra from Im7-6 protein purified from in vitro glycosylation (IVG) reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 UDP-Glc. Full conversion to N -linked glucose was observed after IVG incubation for 24 h at 30°C. (d) Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions assembled from CFPS reaction products with 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30°C. Observed mass shifts and MS/MS fragmentation spectra ( Supplementary Fig. 1 ) are consistent with efficient modification of N -linked glucose with β1-4Gal; β1-4Gal; β1-3GalNAc; and α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Spectra from Hpβ4GalT, Btβ4GalT1, and SpWchJ+K, which did not modify the N -linked glucose installed by ApNGT are shown in Supplementary Fig. 2 . All IVG reactions contained 10 µM Im7 and were incubated for 20 h with 2.5 mM of each appropriate nucleotide-activated sugar donor as indicated above. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n=3 independent IVGs. Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method.

    Techniques Used: In Vitro, Activity Assay, Standard Deviation, Expressing, Mass Spectrometry, Purification, Incubation, Tandem Mass Spectroscopy, Modification

    Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways not containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The sugars installed by NmLgtB, BtGGTA, HpFutA, and HpFutC were susceptible to cleavage by commercially available β1-4 Galactosidase S; α1-3,6 Galactosidase; α1-3,4 Fucosidase; and α1-2 Fucosidase, respectfully. The galactose installed by NmLgtC was resistant to cleavage by β1-4 Galactosidase S and α1-3,6 Galactosidase, but susceptible to cleavage by α1-3,4,6 Galactosidase. The LacNAc polymer installed by alternating activities by NmLgtB and NgLgtA was susceptible to cleavage by a mixture of β1-4 Galactosidase S and the β- N -Acetylglucosaminidase S. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures. Cleavage observations are consistent with previously established GT activities ( Figs. 2 - 3 and Supplementary Table 4 ). See Methods section for exoglycosidase details.
    Figure Legend Snippet: Exoglycosidase sequencing of Im7-6 modified by GlycoPRIME biosynthetic pathways not containing sialic acids. Completed IVG reactions from the GlycoPRIME workflow where purified using Ni-NTA magnetic beads, incubated at 37°C for at least 4 h with and without indicated commercially available exoglycosidases, trypsinized overnight, and then analyzed by glycopeptide LC-MS. The sugars installed by NmLgtB, BtGGTA, HpFutA, and HpFutC were susceptible to cleavage by commercially available β1-4 Galactosidase S; α1-3,6 Galactosidase; α1-3,4 Fucosidase; and α1-2 Fucosidase, respectfully. The galactose installed by NmLgtC was resistant to cleavage by β1-4 Galactosidase S and α1-3,6 Galactosidase, but susceptible to cleavage by α1-3,4,6 Galactosidase. The LacNAc polymer installed by alternating activities by NmLgtB and NgLgtA was susceptible to cleavage by a mixture of β1-4 Galactosidase S and the β- N -Acetylglucosaminidase S. All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated species of the Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK containing an ApNGT glycosylation acceptor sequence. All indicated glycopeptide products are triply charged ions consistent with this Im7-6 tryptic peptide modified with indicated sugar structures. Cleavage observations are consistent with previously established GT activities ( Figs. 2 - 3 and Supplementary Table 4 ). See Methods section for exoglycosidase details.

    Techniques Used: Sequencing, Modification, Purification, Magnetic Beads, Incubation, Liquid Chromatography with Mass Spectroscopy

    Glycopeptide MS/MS spectra of GlycoPRIME reaction products from four and five enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing four and five enzyme pathways modifying Im7-6 shown in Fig. 3d and Supplementary Fig. 12 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3d and Supplementary Fig. 12 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ). Although products from five-enzyme biosynthetic pathway product could not be unambiguous defined, sugar and glycopeptide fragments do suggest modification with both fucose and sialic acids. All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated enzymes, and appropriate sugar donors according to established GT activities.
    Figure Legend Snippet: Glycopeptide MS/MS spectra of GlycoPRIME reaction products from four and five enzyme biosynthetic pathways elaborating N -linked lactose. Products from IVG reactions containing four and five enzyme pathways modifying Im7-6 shown in Fig. 3d and Supplementary Fig. 12 were purified, trypsinized, and analyzed by pseudo MRM MS/MS fragmentation at theoretical glycopeptide masses (indicated by red diamonds) corresponding to detected protein MS peaks in Fig. 3d and Supplementary Fig. 12 . All glycopeptides were fragmented using a collisional energy of 30 eV with a window of ± 2 m/z from targeted m/z values (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. Predicted sugar linkages based on previously established GT activities ( Supplementary Table 4 ). Although products from five-enzyme biosynthetic pathway product could not be unambiguous defined, sugar and glycopeptide fragments do suggest modification with both fucose and sialic acids. All IVG reactions contained Im7-6, ApNGT, NmLgtB, indicated enzymes, and appropriate sugar donors according to established GT activities.

    Techniques Used: Tandem Mass Spectroscopy, Purification, Derivative Assay, Modification

    Glycopeptide MS/MS spectra of GlycoPRIME reaction products from two enzyme biosynthetic pathways elaborating N -linked glucose. Products from IVG reactions containing two enzyme pathways modifying Im7-6 shown in Fig. 2 were purified, trypsinized, and analyzed by pseudo Multiple Reaction Monitoring (MRM) MS/MS fragmentation at theoretical glycopeptide masses (red diamonds) corresponding to detected protein MS peaks using a collisional energy of 30 eV (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. (a) MS/MS spectra of 999.49 ± 2 m/z corresponding to N -linked Glcβ1-3GalNAc installed by BfGalNAcT. (b) MS/MS spectra of 1418.29 ± 2 m/z corresponding to N -linked dextran polymer installed by Apα1-6. (c) MS/MS spectra of 985.81 ± 2 m/z corresponding with N -linked lactose installed by NmLgtB. All IVG reactions contained Im7-6, ApNGT, and appropriate sugar donors according to established enzyme activities ( Supplementary Table 4 ).
    Figure Legend Snippet: Glycopeptide MS/MS spectra of GlycoPRIME reaction products from two enzyme biosynthetic pathways elaborating N -linked glucose. Products from IVG reactions containing two enzyme pathways modifying Im7-6 shown in Fig. 2 were purified, trypsinized, and analyzed by pseudo Multiple Reaction Monitoring (MRM) MS/MS fragmentation at theoretical glycopeptide masses (red diamonds) corresponding to detected protein MS peaks using a collisional energy of 30 eV (see Methods ). Spectra representative of many MS/MS acquisitions from n=1 IVG reaction. Theoretical protein, peptide, and sugar ion masses derived from expected glycosylation structures are shown in Supplementary Tables 3 and 5 . All indicated sugar ions are singly charged and glycopeptide fragmentation products are triply charged ions consistent with modification of Im7-6 tryptic peptide EATTGGNWTTAGGDVLDVLLEHFVK with indicated sugar structures. (a) MS/MS spectra of 999.49 ± 2 m/z corresponding to N -linked Glcβ1-3GalNAc installed by BfGalNAcT. (b) MS/MS spectra of 1418.29 ± 2 m/z corresponding to N -linked dextran polymer installed by Apα1-6. (c) MS/MS spectra of 985.81 ± 2 m/z corresponding with N -linked lactose installed by NmLgtB. All IVG reactions contained Im7-6, ApNGT, and appropriate sugar donors according to established enzyme activities ( Supplementary Table 4 ).

    Techniques Used: Tandem Mass Spectroscopy, Purification, Derivative Assay, Modification

    GlycoPRIME screening of biosynthetic pathways containing five enzymes. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated GTs, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified from and analyzed by intact protein MS. Deconvoluted spectra representative of n=2 IVGs. (a) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 1.63 µM PdST6. (b) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 1.63 µM PdST6 (also shown in Fig. 3d ) (c) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. (d) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. Spectra in a and b as well as fragmentation spectra in Supplementary Fig. 10 indicated three and one species, respectively, which contained both sialic acid and fucose. Predicted glycosylation structures based on previously established GT activities ( Supplementary Table 4 ) and fragmentation spectra ( Supplementary Fig. 10 ). Although structures cannot be unambiguously identified, the previously observed incompatibility of HpFutA and PdST6 as well as the presence of a 1083 m/z peak (Glcβ4Galα6Sia) and the absence of a 1034 m/z (Glc(α3Fuc)β4Gal) peak in fragmentation spectra suggests that in b the proximal galactose is modified with a sialic acid while the GlcNAc is modified with the fucose. No peaks in c or d were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (see Supplementary Table 3 for theoretical mass values).
    Figure Legend Snippet: GlycoPRIME screening of biosynthetic pathways containing five enzymes. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, indicated GTs, and 2.5 mM of appropriate sugar donors (UDP-Glc, UDP-Gal, CMP-Sia, and GDP-Fuc) were purified from and analyzed by intact protein MS. Deconvoluted spectra representative of n=2 IVGs. (a) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 1.63 µM PdST6. (b) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 1.63 µM PdST6 (also shown in Fig. 3d ) (c) Deconvoluted intact protein MS of IVG reactions containing 1.63 µM HpFutA, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. (d) Deconvoluted intact protein MS of IVG reactions containing 0.87 µM HpFutC, 3.83 µM NgLgtA, and 0.43 µM CjCST-I. Spectra in a and b as well as fragmentation spectra in Supplementary Fig. 10 indicated three and one species, respectively, which contained both sialic acid and fucose. Predicted glycosylation structures based on previously established GT activities ( Supplementary Table 4 ) and fragmentation spectra ( Supplementary Fig. 10 ). Although structures cannot be unambiguously identified, the previously observed incompatibility of HpFutA and PdST6 as well as the presence of a 1083 m/z peak (Glcβ4Galα6Sia) and the absence of a 1034 m/z (Glc(α3Fuc)β4Gal) peak in fragmentation spectra suggests that in b the proximal galactose is modified with a sialic acid while the GlcNAc is modified with the fucose. No peaks in c or d were detected that indicated the presence of Im7-6 modified with both a sialic acid and a fucose (see Supplementary Table 3 for theoretical mass values).

    Techniques Used: Purification, Modification

    Deconvoluted intact protein MS spectra of IVG reaction products showing no modification of N -linked glucose installed by ApNGT. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and one elaborating GT were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra of IVG containing 1.3 µM of Hpβ4GalT. (b) Deconvoluted intact protein MS spectra of IVG containing 1.4 µM of Btβ4GalT1 supplemented with 10 µM α-lactalbumin and performed under oxidizing conditions (see Methods ). (c) Deconvoluted intact protein MS spectra of IVG containing 1.5 µM of SpWchJ and 1.0 µM of SpWchK. No peaks were detected that indicated the modification of Im7-6 with N -linked glucose installed by ApNGT (theoretical mass values shown in Supplementary Table 3 ). Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. Deconvoluted spectra shown here are representative of n=2 IVG reactions.
    Figure Legend Snippet: Deconvoluted intact protein MS spectra of IVG reaction products showing no modification of N -linked glucose installed by ApNGT. Products of IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, 2.5 mM of appropriate sugar donors, and one elaborating GT were purified and analyzed by intact protein MS (see Methods ). (a) Deconvoluted intact protein MS spectra of IVG containing 1.3 µM of Hpβ4GalT. (b) Deconvoluted intact protein MS spectra of IVG containing 1.4 µM of Btβ4GalT1 supplemented with 10 µM α-lactalbumin and performed under oxidizing conditions (see Methods ). (c) Deconvoluted intact protein MS spectra of IVG containing 1.5 µM of SpWchJ and 1.0 µM of SpWchK. No peaks were detected that indicated the modification of Im7-6 with N -linked glucose installed by ApNGT (theoretical mass values shown in Supplementary Table 3 ). Spectra from m/z 100-2000 were deconvoluted into 11,000-14,000 Da using Compass Data Analysis maximum entropy method. Deconvoluted spectra shown here are representative of n=2 IVG reactions.

    Techniques Used: Modification, Purification

    20) Product Images from "Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2"

    Article Title: Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16966-3

    HULC interacts with the glycolytic enzyme PKM2. a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads ® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P
    Figure Legend Snippet: HULC interacts with the glycolytic enzyme PKM2. a Biotinylated HULC and antisense HULC were synthesized by in vitro transcription and incubated with HepG2 cell lysates, respectively. The RNA-protein complexes were isolated with streptavidin-conjugated beads. PKM2 in the pull down was examined by western blotting. Biotinylated antisense HULC was used as the control. b The cellular localizations of HULC and PKM2 were analyzed by RNA-FISH combined with immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Immunoprecipitation of PKM1 and PKM2. The left panel shows the immunoblots of PKM1 and PKM2 in the cell lysate and immunoprecipitates. The right panel shows the agarose gel electrophoresis images of the qRT-PCR products of HULC. LincRNA-p21 was examined as RNA control. d Binding of HULC to flag-tagged exon 9 (PKM1 specific) and exon 10 (PKM2 specific) as determined by the RIP assay. e His-tagged rPKM2 was first immobilized to Dynabeads ® His-tag isolation magnetic beads, and then incubated with in vitro transcribed HULC or antisense HULC. The RNA-protein complexes were isolated, and the levels of HULC were examined by qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P

    Techniques Used: Synthesized, In Vitro, Incubation, Isolation, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Immunoprecipitation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Binding Assay, Magnetic Beads

    21) Product Images from "Oligodendroglial Argonaute protein Ago2 associates with molecules of the Mbp mRNA localization machinery and is a downstream target of Fyn kinase"

    Article Title: Oligodendroglial Argonaute protein Ago2 associates with molecules of the Mbp mRNA localization machinery and is a downstream target of Fyn kinase

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2015.00328

    Ago2 associates with the Mbp mRNA transport machinery. (A) FLAG-tagged proteins were immunoprecipitated from FLAG/HA-Ago2 and A2-Myc/His or eGFP overexpressing Oli- neu cells using FLAG-M2 magnetic beads to analyze interaction of Ago2 and hnRNP A2 (lanes 3 and 4). The horizontal line separates two independent blots with equal sample loading. Lanes 1 and 2 show 2.5% of the protein input that was used for the immunoprecipitation (IP). Proteins were analyzed by western blotting using antibodies against HA- and Myc-tag. Antibodies against GAPDH and β-Actin were used to show specificity of the IP. (B) Immunoprecipitation of FLAG-tagged proteins from hnRNP A2b-FLAG stably expressing Oli- neu cells using FLAG-M2 magnetic beads (lane 2). Proteins were analyzed by Western blotting using antibodies against FLAG-tag and Ago2. Antibodies against GAPDH were used to show specificity of the IP. Lane 1 (input) shows 2.5% of the protein input that was used for the IP and lane 3 (unbound) the unbound protein fraction after incubation with the FLAG antibody beads. (C) IP of FLAG-tagged proteins from FLAG/HA-Ago2 or GFP overexpressing Oli- neu cells using FLAG-M2 magnetic beads (FLAG-IP, lanes 3 and 4). Lanes 1 and 2 (input) show 2.5% of the protein input that was used for the IP. Proteins were analyzed by western blotting using antibodies against HA-tag and α-Tubulin to show the specificity of the IP. (D) RNA was extracted from IP eluate of the experiment shown in C and analyzed by RT-PCR for sncRNA715 and Mbp mRNA. PCR products of Mbp (88 nt) and sncRNA715 (∼60 nt, due to the use of hairpin primers) were visualized in an ethidium bromide-stained 4% agarose gel. Positive control for sncRNA715 is the synthetic sncRNA715 (715-mimic) and for Mbp mRNA total RNA from primary rat oligodendrocytes at 14DIV. Negative control (bead control) reveals IP-reaction without addition of protein lysate to ensure specificity of the signals.
    Figure Legend Snippet: Ago2 associates with the Mbp mRNA transport machinery. (A) FLAG-tagged proteins were immunoprecipitated from FLAG/HA-Ago2 and A2-Myc/His or eGFP overexpressing Oli- neu cells using FLAG-M2 magnetic beads to analyze interaction of Ago2 and hnRNP A2 (lanes 3 and 4). The horizontal line separates two independent blots with equal sample loading. Lanes 1 and 2 show 2.5% of the protein input that was used for the immunoprecipitation (IP). Proteins were analyzed by western blotting using antibodies against HA- and Myc-tag. Antibodies against GAPDH and β-Actin were used to show specificity of the IP. (B) Immunoprecipitation of FLAG-tagged proteins from hnRNP A2b-FLAG stably expressing Oli- neu cells using FLAG-M2 magnetic beads (lane 2). Proteins were analyzed by Western blotting using antibodies against FLAG-tag and Ago2. Antibodies against GAPDH were used to show specificity of the IP. Lane 1 (input) shows 2.5% of the protein input that was used for the IP and lane 3 (unbound) the unbound protein fraction after incubation with the FLAG antibody beads. (C) IP of FLAG-tagged proteins from FLAG/HA-Ago2 or GFP overexpressing Oli- neu cells using FLAG-M2 magnetic beads (FLAG-IP, lanes 3 and 4). Lanes 1 and 2 (input) show 2.5% of the protein input that was used for the IP. Proteins were analyzed by western blotting using antibodies against HA-tag and α-Tubulin to show the specificity of the IP. (D) RNA was extracted from IP eluate of the experiment shown in C and analyzed by RT-PCR for sncRNA715 and Mbp mRNA. PCR products of Mbp (88 nt) and sncRNA715 (∼60 nt, due to the use of hairpin primers) were visualized in an ethidium bromide-stained 4% agarose gel. Positive control for sncRNA715 is the synthetic sncRNA715 (715-mimic) and for Mbp mRNA total RNA from primary rat oligodendrocytes at 14DIV. Negative control (bead control) reveals IP-reaction without addition of protein lysate to ensure specificity of the signals.

    Techniques Used: Immunoprecipitation, Magnetic Beads, Western Blot, Stable Transfection, Expressing, FLAG-tag, Incubation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Positive Control, Negative Control

    22) Product Images from "Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex"

    Article Title: Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.809632

    Leading model describing the general architecture of MyoA (motor domain, light purple circle ; converter domain, dark purple oval ), with ELC1 ( orange ) and MLC1 ( teal ) bound to the neck region ( dark purple cylinder ), and accessory proteins comprising the glideosome macromolecular complex.
    Figure Legend Snippet: Leading model describing the general architecture of MyoA (motor domain, light purple circle ; converter domain, dark purple oval ), with ELC1 ( orange ) and MLC1 ( teal ) bound to the neck region ( dark purple cylinder ), and accessory proteins comprising the glideosome macromolecular complex.

    Techniques Used:

    ELC1 binds calcium using a classical EF-hand motif and interacts with MLC1, increasing its affinity for MyoA. a , putative EF-hands 1 and 2 of ELC1, based on consensus sequences. Ca 2+ -interacting residues are numbered , and mutated aspartates are boxed in red. b , bar graphs of representative peptides from ELC1 WT and mutants, showing changes in backbone deuterium exchange induced by Ca 2+ binding. Sequences of peptides are shown above corresponding graphs. The data are from a single time point of exchange (300 s at 23 °C). The error bars for the full HDX-MS data set relating to this figure. c , representative ITC binding isotherms of CaCl 2 titrated into ELC1 wild-type ( left panel ), ELC1 D15A ( middle panel ), or ELC1 D80A ( right panel ). d , representative ITC binding isotherms of ELC1 titrated into MyoA (775–818) without ( left panel ) and with ( right panel ) calcium. e , representative ITC binding isotherms ELC1 titrated into MyoA (775–818), prebound with MLC1 without ( left panel ) or with ( right panel ) calcium.
    Figure Legend Snippet: ELC1 binds calcium using a classical EF-hand motif and interacts with MLC1, increasing its affinity for MyoA. a , putative EF-hands 1 and 2 of ELC1, based on consensus sequences. Ca 2+ -interacting residues are numbered , and mutated aspartates are boxed in red. b , bar graphs of representative peptides from ELC1 WT and mutants, showing changes in backbone deuterium exchange induced by Ca 2+ binding. Sequences of peptides are shown above corresponding graphs. The data are from a single time point of exchange (300 s at 23 °C). The error bars for the full HDX-MS data set relating to this figure. c , representative ITC binding isotherms of CaCl 2 titrated into ELC1 wild-type ( left panel ), ELC1 D15A ( middle panel ), or ELC1 D80A ( right panel ). d , representative ITC binding isotherms of ELC1 titrated into MyoA (775–818) without ( left panel ) and with ( right panel ) calcium. e , representative ITC binding isotherms ELC1 titrated into MyoA (775–818), prebound with MLC1 without ( left panel ) or with ( right panel ) calcium.

    Techniques Used: Binding Assay, Mass Spectrometry

    23) Product Images from "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways"

    Article Title: A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12024-9

    In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. a Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields indicate mean and standard deviation (s.d.) from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 and Supplementary Figs. 1 – 2 . b Symbol key and successful pathways for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. Glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. Sialic acid refers to N -acetylneuraminic acid. c Deconvoluted mass spectrometry spectra from Im7-6 protein purified from IVG reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 mM UDP-Glc. Full conversion to N -linked glucose was observed after 24 h at 30 °C. d Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30 °C. Observed mass shifts and MS/MS fragmentation spectra (Supplementary Fig. 3 ) are consistent with efficient modification of N -linked glucose with β1-4Gal, β1-4Gal, β1-3GalNAc, or α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Hpβ4GalT, Btβ4GalT1, and SpWchJ + K did not modify the N -linked glucose installed by ApNGT (Supplementary Fig. 4 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n = 3 independent IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file
    Figure Legend Snippet: In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. a Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields indicate mean and standard deviation (s.d.) from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 and Supplementary Figs. 1 – 2 . b Symbol key and successful pathways for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. Glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. Sialic acid refers to N -acetylneuraminic acid. c Deconvoluted mass spectrometry spectra from Im7-6 protein purified from IVG reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 mM UDP-Glc. Full conversion to N -linked glucose was observed after 24 h at 30 °C. d Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30 °C. Observed mass shifts and MS/MS fragmentation spectra (Supplementary Fig. 3 ) are consistent with efficient modification of N -linked glucose with β1-4Gal, β1-4Gal, β1-3GalNAc, or α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Hpβ4GalT, Btβ4GalT1, and SpWchJ + K did not modify the N -linked glucose installed by ApNGT (Supplementary Fig. 4 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n = 3 independent IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file

    Techniques Used: In Vitro, Activity Assay, Standard Deviation, Expressing, Mass Spectrometry, Purification, Gas Chromatography, Modification

    In vitro synthesis and assembly of complex glycosylation pathways. a Protein name, species, previously characterized specificity (Supplementary Table 4 ), and optimized CFPS soluble yields (Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields indicate mean and s.d. from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 9 ). ( b ) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, and 2.5 mM appropriate nucleotide-activated sugar donors as well as 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides (Supplementary Fig. 7 ), and exoglycosidase digestions (Supplementary Figs. 10 - 11 ) are consistent with modification of N -linked lactose with α1-3 Gal, α1-4 Gal, α1-3 Fuc, α2-6 Sia, α2-3 Sia, α2-8 Sia, β1-3 GlcNAc, or pyruvylation according to known activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. d Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four- and five- enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors, and indicated GTs at half or one third the concentrations indicated in ( b ) for four- and five- enzyme pathways, respectively. Intact mass shifts and fragmentation spectra (Supplementary Fig. 12 ) are consistent with fucosylation and sialylation of the LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 6 – 8 and 12 – 14 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30 °C, supplemented with an additional 2.5 mM sugar donors and incubated for another 24 h at 30 °C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of n = 2 IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file
    Figure Legend Snippet: In vitro synthesis and assembly of complex glycosylation pathways. a Protein name, species, previously characterized specificity (Supplementary Table 4 ), and optimized CFPS soluble yields (Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields indicate mean and s.d. from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 9 ). ( b ) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, and 2.5 mM appropriate nucleotide-activated sugar donors as well as 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides (Supplementary Fig. 7 ), and exoglycosidase digestions (Supplementary Figs. 10 - 11 ) are consistent with modification of N -linked lactose with α1-3 Gal, α1-4 Gal, α1-3 Fuc, α2-6 Sia, α2-3 Sia, α2-8 Sia, β1-3 GlcNAc, or pyruvylation according to known activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. d Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four- and five- enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors, and indicated GTs at half or one third the concentrations indicated in ( b ) for four- and five- enzyme pathways, respectively. Intact mass shifts and fragmentation spectra (Supplementary Fig. 12 ) are consistent with fucosylation and sialylation of the LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 6 – 8 and 12 – 14 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30 °C, supplemented with an additional 2.5 mM sugar donors and incubated for another 24 h at 30 °C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of n = 2 IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file

    Techniques Used: In Vitro, Mass Spectrometry, Purification, Modification, Produced, Incubation

    24) Product Images from "Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization"

    Article Title: Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0491-9

    Anti- Campylobacter nanobodies interact with native outer membrane proteins. Serial tenfold dilutions of the nanobodies were used in ELISA to assess the binding with linear or conformational epitopes. OMPs (1 µg/mL) were coated in a 96-well plate and the interaction of His-tagged nanobodies with native, untreated OMP, and with denatured protein extract was measured. Binding of A Nb5, B Nb22, C Nb23, D Nb24, E Nb49 and F Nb84 was measured. For detection, mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase were used. The error bars represent the standard deviations.
    Figure Legend Snippet: Anti- Campylobacter nanobodies interact with native outer membrane proteins. Serial tenfold dilutions of the nanobodies were used in ELISA to assess the binding with linear or conformational epitopes. OMPs (1 µg/mL) were coated in a 96-well plate and the interaction of His-tagged nanobodies with native, untreated OMP, and with denatured protein extract was measured. Binding of A Nb5, B Nb22, C Nb23, D Nb24, E Nb49 and F Nb84 was measured. For detection, mouse anti-Histidine tag monoclonal antibody and goat anti-mouse IgG conjugated to alkaline phosphatase were used. The error bars represent the standard deviations.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.
    Figure Legend Snippet: Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.

    Techniques Used: Agglutination, Negative Control, Microscopy

    Amino acid sequence alignment of anti- Campylobacter nanobodies selected for their broad specificity. The structural framework regions are indicated by FR1–FR4 and the red boxes specify the CDRs. On the basis of the variation of the amino acid sequence of the CDR3, the nanobodies were divided in twelve unique groups.
    Figure Legend Snippet: Amino acid sequence alignment of anti- Campylobacter nanobodies selected for their broad specificity. The structural framework regions are indicated by FR1–FR4 and the red boxes specify the CDRs. On the basis of the variation of the amino acid sequence of the CDR3, the nanobodies were divided in twelve unique groups.

    Techniques Used: Sequencing

    Detection of the interaction of anti- Campylobacter nanobodies and C. jejuni KC40 by immunofluorescence microscopy. The interaction was detected by ( A , C , E , G ) immunofluorescence microscopy and the C. jejuni cells ( B , D , F , H ) were visualised by bright field microscopy. A , B A nanobody specific for F4-fimbriated enterotoxigenic E. coli shows no binding with the C. jejuni cells. C , D ; E , F and G , H The anti- Campylobacter nanobodies Nb22, Nb23 and Nb84 respectively, binds specifically with the C. jejuni cells.
    Figure Legend Snippet: Detection of the interaction of anti- Campylobacter nanobodies and C. jejuni KC40 by immunofluorescence microscopy. The interaction was detected by ( A , C , E , G ) immunofluorescence microscopy and the C. jejuni cells ( B , D , F , H ) were visualised by bright field microscopy. A , B A nanobody specific for F4-fimbriated enterotoxigenic E. coli shows no binding with the C. jejuni cells. C , D ; E , F and G , H The anti- Campylobacter nanobodies Nb22, Nb23 and Nb84 respectively, binds specifically with the C. jejuni cells.

    Techniques Used: Immunofluorescence, Microscopy, Binding Assay

    25) Product Images from "Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization"

    Article Title: Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0491-9

    Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.
    Figure Legend Snippet: Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.

    Techniques Used: Agglutination, Negative Control, Microscopy

    26) Product Images from "The Hsp70 co-chaperone Ydj1/HDJ2 regulates ribonucleotide reductase activity"

    Article Title: The Hsp70 co-chaperone Ydj1/HDJ2 regulates ribonucleotide reductase activity

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007462

    RNR interacts with Hsp40 in yeast and mammalian cells. (A) Rnr2 interacts with Ydj1 in yeast. WT cells transformed with either pRS313 or plasmid expressing FLAG-tagged Rnr2 were grown to exponential phase and were either left untreated or were treated with HU as in Fig 3 . Cell extracts (lysate) and immunoprecipitates (IP) with anti-FLAG M2 magnetic beads were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibodies to detect Rnr2 or anti-Ydj1 antibodies to detect Ydj1. (B) R2B interacts with HDJ2 in mammalian cells. HEK293 cells were transfected with a plasmid expressing CMV-driven HIS 6 -tagged R2B. Cells extracts were obtained 48 hours post-transfection. Cell extracts (lysate) and immunoprecipitates (IP) with HIS-dynabeads were subjected to SDS-PAGE and analyzed by immunoblotting with tetra-HIS antibodies to detect R2B or anti-HDJ2 antibodies to detect HDJ2.
    Figure Legend Snippet: RNR interacts with Hsp40 in yeast and mammalian cells. (A) Rnr2 interacts with Ydj1 in yeast. WT cells transformed with either pRS313 or plasmid expressing FLAG-tagged Rnr2 were grown to exponential phase and were either left untreated or were treated with HU as in Fig 3 . Cell extracts (lysate) and immunoprecipitates (IP) with anti-FLAG M2 magnetic beads were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibodies to detect Rnr2 or anti-Ydj1 antibodies to detect Ydj1. (B) R2B interacts with HDJ2 in mammalian cells. HEK293 cells were transfected with a plasmid expressing CMV-driven HIS 6 -tagged R2B. Cells extracts were obtained 48 hours post-transfection. Cell extracts (lysate) and immunoprecipitates (IP) with HIS-dynabeads were subjected to SDS-PAGE and analyzed by immunoblotting with tetra-HIS antibodies to detect R2B or anti-HDJ2 antibodies to detect HDJ2.

    Techniques Used: Transformation Assay, Plasmid Preparation, Expressing, Magnetic Beads, SDS Page, Transfection

    27) Product Images from "Mutational Analysis of Trypanosoma brucei RNA Editing Ligase Reveals Regions Critical for Interaction with KREPA2"

    Article Title: Mutational Analysis of Trypanosoma brucei RNA Editing Ligase Reveals Regions Critical for Interaction with KREPA2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120844

    Three Tb REL1 truncation mutants. A) Schematic representation of full length Tb REL1 and the three truncation mutants. (1) Tb REL1 full length, showing the five motifs (I–V) common to all members of the nucleotidyl transferase family, along with R372 and the DALKD motif (2) Tb REL1-N term, containing the five signature motifs critical for catalysis. (3) Tb REL1-R372, containing the five N-terminal motifs and R372 (4) Tb REL1-DALKD, containing the DALKD motif and lacking the final 59 amino acids. (B). Recombinant Tb REL1 truncation mutants were expressed, precipitated using His-tag isolation beads and separated by 10% SDS-PAGE. (C) Co-precipitation of KREPA2 with full-length Tb REL1 and truncation mutants. A KREPA2 only control was run as a negative control to check for background binding of KREPA2 to the His-tag isolation beads. All truncation mutants were unable to pull down KREPA2.
    Figure Legend Snippet: Three Tb REL1 truncation mutants. A) Schematic representation of full length Tb REL1 and the three truncation mutants. (1) Tb REL1 full length, showing the five motifs (I–V) common to all members of the nucleotidyl transferase family, along with R372 and the DALKD motif (2) Tb REL1-N term, containing the five signature motifs critical for catalysis. (3) Tb REL1-R372, containing the five N-terminal motifs and R372 (4) Tb REL1-DALKD, containing the DALKD motif and lacking the final 59 amino acids. (B). Recombinant Tb REL1 truncation mutants were expressed, precipitated using His-tag isolation beads and separated by 10% SDS-PAGE. (C) Co-precipitation of KREPA2 with full-length Tb REL1 and truncation mutants. A KREPA2 only control was run as a negative control to check for background binding of KREPA2 to the His-tag isolation beads. All truncation mutants were unable to pull down KREPA2.

    Techniques Used: Recombinant, Isolation, SDS Page, Negative Control, Binding Assay

    Expression of Tb REL1 and KREPA2. (A) Both proteins were in vitro transcribed/translated in the presence of [ 35 S]-methionine and separated by 10% SDS-PAGE. Tb REL1 (52 kDa) and KREPA2 (63 kDa) migrated at the expected size. (B) Western blot analysis of His-tagged Tb REL1. The blot shows 0.3125, 0.625, 1.25, 2.5, and 5 μL of a 6x His protein ladder of known amounts (3.125, 6.25, 12.5, 25 and 50 ng corresponding to the 50 KDa protein) along with 2 μL of purified Tb REL1 WT and mutation E81A.
    Figure Legend Snippet: Expression of Tb REL1 and KREPA2. (A) Both proteins were in vitro transcribed/translated in the presence of [ 35 S]-methionine and separated by 10% SDS-PAGE. Tb REL1 (52 kDa) and KREPA2 (63 kDa) migrated at the expected size. (B) Western blot analysis of His-tagged Tb REL1. The blot shows 0.3125, 0.625, 1.25, 2.5, and 5 μL of a 6x His protein ladder of known amounts (3.125, 6.25, 12.5, 25 and 50 ng corresponding to the 50 KDa protein) along with 2 μL of purified Tb REL1 WT and mutation E81A.

    Techniques Used: Expressing, In Vitro, SDS Page, Western Blot, Purification, Mutagenesis

    KREPA2 stimulates Tb REL1 activity. (A) Stimulatory effect of KREPA2 on Tb REL1 adenylylation. A graph representing the adenylylation enhancement (6.7 fold) achieved by adding KREPA2 is seen below. Here, the X-axis represents the adenylylation activity (arbitrary values obtained from Quantity One volume measurement tool). The error bars represent standard deviation between triplicate samples. (B) Stimulatory effect of KREPA2 on Tb REL1-mediated ligation. Two minor bands (single star) are a result of nonspecific ligation of input degradation products. The illustration shows the input 5′ [α- 32 P]-GTP-capped RNA fragment along with the unlabeled 3′ fragment and gRNA, and the [α- 32 P]-GTP-capped ligated product. A graph representing the stimulation of ligation achieved by adding KREPA2 (3.6 fold) is seen below. Here, the X-axis represents the ligation activity (arbitrary values obtained from Quantity One volume measurement tool). The error bars represent standard deviation between triplicate samples.
    Figure Legend Snippet: KREPA2 stimulates Tb REL1 activity. (A) Stimulatory effect of KREPA2 on Tb REL1 adenylylation. A graph representing the adenylylation enhancement (6.7 fold) achieved by adding KREPA2 is seen below. Here, the X-axis represents the adenylylation activity (arbitrary values obtained from Quantity One volume measurement tool). The error bars represent standard deviation between triplicate samples. (B) Stimulatory effect of KREPA2 on Tb REL1-mediated ligation. Two minor bands (single star) are a result of nonspecific ligation of input degradation products. The illustration shows the input 5′ [α- 32 P]-GTP-capped RNA fragment along with the unlabeled 3′ fragment and gRNA, and the [α- 32 P]-GTP-capped ligated product. A graph representing the stimulation of ligation achieved by adding KREPA2 (3.6 fold) is seen below. Here, the X-axis represents the ligation activity (arbitrary values obtained from Quantity One volume measurement tool). The error bars represent standard deviation between triplicate samples.

    Techniques Used: Activity Assay, Standard Deviation, Ligation

    Multiple sequence alignment between kinetoplastid RNA editing ligases and T4 RNA Ligase 2. The amino acid sequences of Tb REL1 and Tb REL2 from Trypanosoma brucei were aligned with sequences from the corresponding ligases from Trypanosoma cruzi , Leishmania major , and T4 bacteriophage. Nucleotidyl transferase motifs I–V are shown in shaded boxes. The hydrophobic loop H9 and DALKD motif are highlighted in yellow and pink. The 17 point mutations used in the present study are labeled above the corresponding wild-type residues in black, and the 3 control point mutations are labeled in grey.
    Figure Legend Snippet: Multiple sequence alignment between kinetoplastid RNA editing ligases and T4 RNA Ligase 2. The amino acid sequences of Tb REL1 and Tb REL2 from Trypanosoma brucei were aligned with sequences from the corresponding ligases from Trypanosoma cruzi , Leishmania major , and T4 bacteriophage. Nucleotidyl transferase motifs I–V are shown in shaded boxes. The hydrophobic loop H9 and DALKD motif are highlighted in yellow and pink. The 17 point mutations used in the present study are labeled above the corresponding wild-type residues in black, and the 3 control point mutations are labeled in grey.

    Techniques Used: Sequencing, Labeling

    Interaction of Tb REL1WT and point mutants with KREPA2. (A) All mutants were expressed and precipitated in the absence and presence of KREAP2. KREPA2 was precipitated alone as a negative control to check for background binding to the his-tag isolation beads (last lane). (B) Graphical representation of the precipitation experiment. The amount of KREPA2 pulled down was first normalized with its own Tb REL1, after correcting for the background binding of KREPA2. Finally, all pull-down values were normalized to the amount of KREPA2 pulled down by Tb REL1 WT (100%). The amount of KREPA2 pulled down by Tb REL1 F206A, K441A, and E444A were less than 50% of the amount pulled down by the Tb REL1 WT. While the X-axis represents Tb REL1 WT and the different point mutants, the Y-axis represents relative pulldown (%). The error bars represent standard deviation between triplicate samples.
    Figure Legend Snippet: Interaction of Tb REL1WT and point mutants with KREPA2. (A) All mutants were expressed and precipitated in the absence and presence of KREAP2. KREPA2 was precipitated alone as a negative control to check for background binding to the his-tag isolation beads (last lane). (B) Graphical representation of the precipitation experiment. The amount of KREPA2 pulled down was first normalized with its own Tb REL1, after correcting for the background binding of KREPA2. Finally, all pull-down values were normalized to the amount of KREPA2 pulled down by Tb REL1 WT (100%). The amount of KREPA2 pulled down by Tb REL1 F206A, K441A, and E444A were less than 50% of the amount pulled down by the Tb REL1 WT. While the X-axis represents Tb REL1 WT and the different point mutants, the Y-axis represents relative pulldown (%). The error bars represent standard deviation between triplicate samples.

    Techniques Used: Negative Control, Binding Assay, Isolation, Standard Deviation

    Graphical representation of the overall effect of KREPA2 on Tb REL1 WT and point mutants. The activity of Tb REL1 is segregated in two domains: N-terminal and C-terminal domains. Mutations in N-terminal domains lead to a considerable loss in adenylylation activity of Tb REL1, which are not rescued by the addition of KREPA2. While ligation activity of some of these point mutations are rescued (E81A, E119A and H205A; yellow bars), they remain affected for the others. Point mutations at F206, T264 and Y275 (shown in red bars) represent residues with severe effects on Tb REL1 enzymatic activity, with F206A having an effect on KREPA2 pull-down as well. While the overall activity of all Tb REL1 point mutants are affected in the C-terminal region, addition of KREPA2 completely rescues point mutations at K379, K405, E410 and W442 (shown in green bars), while having no effect or partially rescuing point mutations at the other residues, K424, K435, K441, K443, E444 and E455.
    Figure Legend Snippet: Graphical representation of the overall effect of KREPA2 on Tb REL1 WT and point mutants. The activity of Tb REL1 is segregated in two domains: N-terminal and C-terminal domains. Mutations in N-terminal domains lead to a considerable loss in adenylylation activity of Tb REL1, which are not rescued by the addition of KREPA2. While ligation activity of some of these point mutations are rescued (E81A, E119A and H205A; yellow bars), they remain affected for the others. Point mutations at F206, T264 and Y275 (shown in red bars) represent residues with severe effects on Tb REL1 enzymatic activity, with F206A having an effect on KREPA2 pull-down as well. While the overall activity of all Tb REL1 point mutants are affected in the C-terminal region, addition of KREPA2 completely rescues point mutations at K379, K405, E410 and W442 (shown in green bars), while having no effect or partially rescuing point mutations at the other residues, K424, K435, K441, K443, E444 and E455.

    Techniques Used: Activity Assay, Ligation

    Ligation efficiency of Tb REL1 WT and point mutants in the absence and presence of KREPA2. (A) Ligation gel images of Tb REL1 WT and point mutants in the absence (top) and presence (bottom) of KREPA2. The intensity of the gel above was increased so that Tb REL1 WT ligation efficiencies could be visually perceived. The faint band seen above the ligated product is an artifact achieved from ligation occurring between 5’lig (16 bases) and glig (36 bases), instead of 3’lig (34 bases). (B) Graphical representation of ligation experiment. The intensity of each mutant in the top gel was normalized to its WT control, and the intensity of each mutant in the bottom gel was normalized to its WT + KREPA2 control. While the X-axis represents Tb REL1 WT and the different point mutants, the Y-axis represents relative ligation activity (%). The error bars represent standard deviation between triplicate samples.
    Figure Legend Snippet: Ligation efficiency of Tb REL1 WT and point mutants in the absence and presence of KREPA2. (A) Ligation gel images of Tb REL1 WT and point mutants in the absence (top) and presence (bottom) of KREPA2. The intensity of the gel above was increased so that Tb REL1 WT ligation efficiencies could be visually perceived. The faint band seen above the ligated product is an artifact achieved from ligation occurring between 5’lig (16 bases) and glig (36 bases), instead of 3’lig (34 bases). (B) Graphical representation of ligation experiment. The intensity of each mutant in the top gel was normalized to its WT control, and the intensity of each mutant in the bottom gel was normalized to its WT + KREPA2 control. While the X-axis represents Tb REL1 WT and the different point mutants, the Y-axis represents relative ligation activity (%). The error bars represent standard deviation between triplicate samples.

    Techniques Used: Ligation, Mutagenesis, Activity Assay, Standard Deviation

    Adenylylation of Tb REL1 WT and point mutants in the absence and presence of KREPA2. (A) Adenylylation gel images of Tb REL1 WT and point mutants in the absence (top) and presence (bottom) of KREPA2. The intensity of the gel above was increased so that Tb REL1 WT adenylylated band intensities matched in both gels (with and without KREPA2), for the purpose of normalization. This gives a better visual perspective on the effect of each mutation with respect to the WT. (B) Graphical representation of adenylylation experiment. The intensity of each mutant in the top gel was normalized to its WT control, and the intensity of each mutant in the bottom gel was normalized to its WT + KREPA2 control. While the X-axis represents Tb REL1 WT and the different point mutants, the Y-axis represents relative adenylylation activity (%). The error bars represent standard deviation between triplicate samples.
    Figure Legend Snippet: Adenylylation of Tb REL1 WT and point mutants in the absence and presence of KREPA2. (A) Adenylylation gel images of Tb REL1 WT and point mutants in the absence (top) and presence (bottom) of KREPA2. The intensity of the gel above was increased so that Tb REL1 WT adenylylated band intensities matched in both gels (with and without KREPA2), for the purpose of normalization. This gives a better visual perspective on the effect of each mutation with respect to the WT. (B) Graphical representation of adenylylation experiment. The intensity of each mutant in the top gel was normalized to its WT control, and the intensity of each mutant in the bottom gel was normalized to its WT + KREPA2 control. While the X-axis represents Tb REL1 WT and the different point mutants, the Y-axis represents relative adenylylation activity (%). The error bars represent standard deviation between triplicate samples.

    Techniques Used: Mutagenesis, Activity Assay, Standard Deviation

    28) Product Images from "A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3"

    Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

    Journal: mAbs

    doi: 10.1080/19420862.2018.1445451

    Purification of recombinant Kv1.3. a. SDS-PAGE analysis of purified and reconstituted Kv1.3. Kv1.3 was purified as described in Materials, resolved by SDS-PAGE before and after reconstitution into liposomes and stained with SimplyBlue™ SafeStain™. B. Ligand Binding Analysis. Kv1.3-containing liposomes were incubated with FAM-ShK (3 nM) in the presence or absence of a 50-fold excess of either MgTx or IbTx. Top Panel is a representative experiment showing total binding expressed as Anisotropy measured by fluorescence polarization. Bottom Panel represents specific binding to FAM-ShK. Kd was estimated as 11.5 nM +/− 3.4 nM based on specific binding curves generated in three separate experiments c. Fluorescence microscopy analysis of Kv1.3 magnetic beads. Magnetic beads containing tethered Kv1.3 reconstituted into a lipid bilayer consisting of rhodamine-labeled PE and non-labeled PC were examined by fluorescence microscopy. Beads were examined under a rhodamine filter to detect labeled PE incorporation (Left Panel) and with a FITC-filter following labelling with an anti-Kv1.3 antibody that recognizes an epitope on the first extracellular loop and anti-guinea pig conjugated FITC (Middle Panel). Rhodamine and FITC- images were merged (Right Panel) to confirm co-localization of Kv1.3 and the lipid bilayer (yellow fluorescence). D. Schematic illustration of Kv1.3 magnetic beads. Shown is the magnetic bead surface; the lipid bilayer consisting of PC (yellow lipids) and Rhodamine-labeled PE (red lipids); the six transmembrane domains of the Kv1.3 monomer (S1-S6); the C-terminal engineered FLAG (orange Triangle) and 10Xhis (Green Box) tags; a star indicates the position of the epitope on the first extracellular loop that is recognized by the Kv1.3 antibody utilized in c and e. e. Kv1.3 magnetic beads preferentially precipitate an antibody that recognizes an extracellular epitope. Kv1.3 magnetic beads or control beads were incubated with antibodies (6.7 nM) that recognize either internal (FLAG and 10Xhis) or external (Kv1.3) epitopes. Beads were washed and bound IgG eluted directly in SDS-PAGE loading buffer. IgG was detected by Western analysis using either anti-mouse-HRP (anti-FLAG and -His) or anti-guinea pig-HRP (anti-Kv1.3).
    Figure Legend Snippet: Purification of recombinant Kv1.3. a. SDS-PAGE analysis of purified and reconstituted Kv1.3. Kv1.3 was purified as described in Materials, resolved by SDS-PAGE before and after reconstitution into liposomes and stained with SimplyBlue™ SafeStain™. B. Ligand Binding Analysis. Kv1.3-containing liposomes were incubated with FAM-ShK (3 nM) in the presence or absence of a 50-fold excess of either MgTx or IbTx. Top Panel is a representative experiment showing total binding expressed as Anisotropy measured by fluorescence polarization. Bottom Panel represents specific binding to FAM-ShK. Kd was estimated as 11.5 nM +/− 3.4 nM based on specific binding curves generated in three separate experiments c. Fluorescence microscopy analysis of Kv1.3 magnetic beads. Magnetic beads containing tethered Kv1.3 reconstituted into a lipid bilayer consisting of rhodamine-labeled PE and non-labeled PC were examined by fluorescence microscopy. Beads were examined under a rhodamine filter to detect labeled PE incorporation (Left Panel) and with a FITC-filter following labelling with an anti-Kv1.3 antibody that recognizes an epitope on the first extracellular loop and anti-guinea pig conjugated FITC (Middle Panel). Rhodamine and FITC- images were merged (Right Panel) to confirm co-localization of Kv1.3 and the lipid bilayer (yellow fluorescence). D. Schematic illustration of Kv1.3 magnetic beads. Shown is the magnetic bead surface; the lipid bilayer consisting of PC (yellow lipids) and Rhodamine-labeled PE (red lipids); the six transmembrane domains of the Kv1.3 monomer (S1-S6); the C-terminal engineered FLAG (orange Triangle) and 10Xhis (Green Box) tags; a star indicates the position of the epitope on the first extracellular loop that is recognized by the Kv1.3 antibody utilized in c and e. e. Kv1.3 magnetic beads preferentially precipitate an antibody that recognizes an extracellular epitope. Kv1.3 magnetic beads or control beads were incubated with antibodies (6.7 nM) that recognize either internal (FLAG and 10Xhis) or external (Kv1.3) epitopes. Beads were washed and bound IgG eluted directly in SDS-PAGE loading buffer. IgG was detected by Western analysis using either anti-mouse-HRP (anti-FLAG and -His) or anti-guinea pig-HRP (anti-Kv1.3).

    Techniques Used: Purification, Recombinant, SDS Page, Staining, Ligand Binding Assay, Incubation, Binding Assay, Fluorescence, Generated, Microscopy, Magnetic Beads, Labeling, Western Blot

    Kv1.3 epitope binning analysis. A. Binning analysis heat map. Shown are antibodies colored by bin (1–5) and by functional data. Antibodies that block Kv1.3 current are highlighted in blue (Ephys only), those that additionally bind Jurkat cells are highlighted in red (Ephys + Jurkat) and those that do neither are highlighted in grey. Relative competition activity of each antibody is color coded and indicates strong competition (red boxes), intermediate/weak competition (yellow boxes) or no competition (green boxes). Dark red boxes indicate competition from the same antibody pair. B. Binning network plot. Antibodies are colored to identify those that inhibit channel activity (blue), additionally bind Jurkat cells (red) or do neither (grey). Note antibody L1A3 was not tested for its ability to bind Jurkat cells, however, for simplicity was denoted as inhibiting ion channel only (blue) in a b.
    Figure Legend Snippet: Kv1.3 epitope binning analysis. A. Binning analysis heat map. Shown are antibodies colored by bin (1–5) and by functional data. Antibodies that block Kv1.3 current are highlighted in blue (Ephys only), those that additionally bind Jurkat cells are highlighted in red (Ephys + Jurkat) and those that do neither are highlighted in grey. Relative competition activity of each antibody is color coded and indicates strong competition (red boxes), intermediate/weak competition (yellow boxes) or no competition (green boxes). Dark red boxes indicate competition from the same antibody pair. B. Binning network plot. Antibodies are colored to identify those that inhibit channel activity (blue), additionally bind Jurkat cells (red) or do neither (grey). Note antibody L1A3 was not tested for its ability to bind Jurkat cells, however, for simplicity was denoted as inhibiting ion channel only (blue) in a b.

    Techniques Used: Functional Assay, Blocking Assay, Activity Assay

    Identification of anti-Kv1.3 antibodies that functionally inhibit Kv1.3 channel activity. Purified scFv-Fc anti-Kv1.3 antibodies from either chickens or llamas were tested at a concentration of 400nM via electrophysiology for their ability to block current from human Kv1.3 expressed in L929 fibroblast cells. Shown are representative traces for each of the antibodies that blocked Kv1.3 current. Black lines represent control currents, red lines represent currents following addition of antibody. Inhibiting anti-Kv1.3 antibody clones derived from chickens are shown in a, the functional llama anti-Kv1.3 antibody is shown in b. An example of an antibody that was tested and shown not to modulate Kv1.3 activity is shown in c. d. Time-dependent development of current inhibition by monoclonal antibodies targeting Kv1.3. (Left Panel) Time-current plots showing current inhibition of three (3) individual cells expressing hKv1.3 channels by the monoclonal antibody ScFv-Fc L1A3. Antibodies were added after current stabilization at 0 second. Currents were elicited by pulsing to +40 mV for 200 ms from a holding potential of −80 mV every 30 seconds. (Right Panel) Means ± SD plot of the current inhibition of three individual cells in the left panel. Similar time-dependent profiles were observed for each of the blocking antibodies.
    Figure Legend Snippet: Identification of anti-Kv1.3 antibodies that functionally inhibit Kv1.3 channel activity. Purified scFv-Fc anti-Kv1.3 antibodies from either chickens or llamas were tested at a concentration of 400nM via electrophysiology for their ability to block current from human Kv1.3 expressed in L929 fibroblast cells. Shown are representative traces for each of the antibodies that blocked Kv1.3 current. Black lines represent control currents, red lines represent currents following addition of antibody. Inhibiting anti-Kv1.3 antibody clones derived from chickens are shown in a, the functional llama anti-Kv1.3 antibody is shown in b. An example of an antibody that was tested and shown not to modulate Kv1.3 activity is shown in c. d. Time-dependent development of current inhibition by monoclonal antibodies targeting Kv1.3. (Left Panel) Time-current plots showing current inhibition of three (3) individual cells expressing hKv1.3 channels by the monoclonal antibody ScFv-Fc L1A3. Antibodies were added after current stabilization at 0 second. Currents were elicited by pulsing to +40 mV for 200 ms from a holding potential of −80 mV every 30 seconds. (Right Panel) Means ± SD plot of the current inhibition of three individual cells in the left panel. Similar time-dependent profiles were observed for each of the blocking antibodies.

    Techniques Used: Activity Assay, Purification, Concentration Assay, Blocking Assay, Clone Assay, Derivative Assay, Functional Assay, Inhibition, Expressing, Mass Spectrometry

    Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.
    Figure Legend Snippet: Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.

    Techniques Used: Expressing, Construct, Modification, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Isolation, Negative Control, Purification, Incubation, Generated, SDS Page, Labeling, Fluorescence, Confocal Microscopy, Binding Assay

    Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.
    Figure Legend Snippet: Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Generated, Activity Assay, Binding Assay

    Anti-Kv1.3 antibody potency analysis. A. Dose response curves. Ten-fold serial dilutions of chicken antibody p1E6 (red traces) and llama antibody L1A3 (green traces) were used to generate dose-response curves. Black lines represent control currents. B. IC 50 Determinations. IC 50 s for p1E6 (red curve) and L1A3 (green curve) were determined by fitting the calculated percentage of current block to a Hill equation. IC 50 s for p1E6 and L1A3 were estimated as 6 and 109 nM, respectively c. Analysis of p1E6 and L1A3 selectivity. ScFv-Fc clones p1E6 and L1A3 were tested for their ability to block the activity of related Kv1.x family members (Kv1.1, Kv1.2, Kv1.5), hERG and Nav1.5 at a concentration of 1 μM. Shown are representative traces from each experiment. Black lines, control current; Red lines, currents following addition of antibody.
    Figure Legend Snippet: Anti-Kv1.3 antibody potency analysis. A. Dose response curves. Ten-fold serial dilutions of chicken antibody p1E6 (red traces) and llama antibody L1A3 (green traces) were used to generate dose-response curves. Black lines represent control currents. B. IC 50 Determinations. IC 50 s for p1E6 (red curve) and L1A3 (green curve) were determined by fitting the calculated percentage of current block to a Hill equation. IC 50 s for p1E6 and L1A3 were estimated as 6 and 109 nM, respectively c. Analysis of p1E6 and L1A3 selectivity. ScFv-Fc clones p1E6 and L1A3 were tested for their ability to block the activity of related Kv1.x family members (Kv1.1, Kv1.2, Kv1.5), hERG and Nav1.5 at a concentration of 1 μM. Shown are representative traces from each experiment. Black lines, control current; Red lines, currents following addition of antibody.

    Techniques Used: Blocking Assay, Clone Assay, Activity Assay, Concentration Assay

    29) Product Images from "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways"

    Article Title: A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12024-9

    In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. a Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields indicate mean and standard deviation (s.d.) from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 and Supplementary Figs. 1 – 2 . b Symbol key and successful pathways for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. Glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. Sialic acid refers to N -acetylneuraminic acid. c Deconvoluted mass spectrometry spectra from Im7-6 protein purified from IVG reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 mM UDP-Glc. Full conversion to N -linked glucose was observed after 24 h at 30 °C. d Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30 °C. Observed mass shifts and MS/MS fragmentation spectra (Supplementary Fig. 3 ) are consistent with efficient modification of N -linked glucose with β1-4Gal, β1-4Gal, β1-3GalNAc, or α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Hpβ4GalT, Btβ4GalT1, and SpWchJ + K did not modify the N -linked glucose installed by ApNGT (Supplementary Fig. 4 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n = 3 independent IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file
    Figure Legend Snippet: In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. a Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4 . CFPS yields indicate mean and standard deviation (s.d.) from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 and Supplementary Figs. 1 – 2 . b Symbol key and successful pathways for N -linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. Glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. Sialic acid refers to N -acetylneuraminic acid. c Deconvoluted mass spectrometry spectra from Im7-6 protein purified from IVG reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 mM UDP-Glc. Full conversion to N -linked glucose was observed after 24 h at 30 °C. d Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30 °C. Observed mass shifts and MS/MS fragmentation spectra (Supplementary Fig. 3 ) are consistent with efficient modification of N -linked glucose with β1-4Gal, β1-4Gal, β1-3GalNAc, or α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3 . Hpβ4GalT, Btβ4GalT1, and SpWchJ + K did not modify the N -linked glucose installed by ApNGT (Supplementary Fig. 4 ). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n = 3 independent IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file

    Techniques Used: In Vitro, Activity Assay, Standard Deviation, Expressing, Mass Spectrometry, Purification, Gas Chromatography, Modification

    In vitro synthesis and assembly of complex glycosylation pathways. a Protein name, species, previously characterized specificity (Supplementary Table 4 ), and optimized CFPS soluble yields (Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields indicate mean and s.d. from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 9 ). ( b ) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, and 2.5 mM appropriate nucleotide-activated sugar donors as well as 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides (Supplementary Fig. 7 ), and exoglycosidase digestions (Supplementary Figs. 10 - 11 ) are consistent with modification of N -linked lactose with α1-3 Gal, α1-4 Gal, α1-3 Fuc, α2-6 Sia, α2-3 Sia, α2-8 Sia, β1-3 GlcNAc, or pyruvylation according to known activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. d Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four- and five- enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors, and indicated GTs at half or one third the concentrations indicated in ( b ) for four- and five- enzyme pathways, respectively. Intact mass shifts and fragmentation spectra (Supplementary Fig. 12 ) are consistent with fucosylation and sialylation of the LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 6 – 8 and 12 – 14 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30 °C, supplemented with an additional 2.5 mM sugar donors and incubated for another 24 h at 30 °C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of n = 2 IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file
    Figure Legend Snippet: In vitro synthesis and assembly of complex glycosylation pathways. a Protein name, species, previously characterized specificity (Supplementary Table 4 ), and optimized CFPS soluble yields (Supplementary Table 2 ) for enzymes tested for elaboration of N -linked lactose. CFPS yields indicate mean and s.d. from n = 3 CFPS reactions quantified by [ 14 C]-leucine incorporation. CjCST-I and HsSIAT1 yields were measured under oxidizing conditions (see Supplementary Fig. 9 ). ( b ) Intact deconvoluted MS spectra from Im7-6 protein purified from IVG reactions with 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, and 2.5 mM appropriate nucleotide-activated sugar donors as well as 4.0 µM BtGGTA, 5.3 µM NmLgtC, 4.9 µM HpFutA, 2.6 µM HpFutC, 4.9 µM PdST6, 5.0 µM CjCST-II, 1.3 µM CjCST-I, 11.5 µM NgLgtA, or 2.2 µM SpPvg1. Mass shifts of intact Im7-6, fragmentation spectra of trypsinized Im7-6 glycopeptides (Supplementary Fig. 7 ), and exoglycosidase digestions (Supplementary Figs. 10 - 11 ) are consistent with modification of N -linked lactose with α1-3 Gal, α1-4 Gal, α1-3 Fuc, α2-6 Sia, α2-3 Sia, α2-8 Sia, β1-3 GlcNAc, or pyruvylation according to known activities of BtGGTA, NmLgtC, HpFutA, HpFutC, PdST6, CjCST-II, CjCST-I, NgLgtA, or SpPvg1. d Deconvoluted intact Im7-6 spectra of fucosylated and sialylated LacNAc structures produced by four- and five- enzyme combinations. IVG reactions contained 10 µM Im7-6, 0.4 µM ApNGT, 2 µM NmLgtB, appropriate sugar donors, and indicated GTs at half or one third the concentrations indicated in ( b ) for four- and five- enzyme pathways, respectively. Intact mass shifts and fragmentation spectra (Supplementary Fig. 12 ) are consistent with fucosylation and sialylation of the LacNAc core according to known activities. Intact protein and glycopeptide fragmentation spectra from other screened GTs and GT combinations not shown here are found in Supplementary Figs. 6 – 8 and 12 – 14 . To provide maximum conversion, IVG reactions were incubated for 24 h at 30 °C, supplemented with an additional 2.5 mM sugar donors and incubated for another 24 h at 30 °C. Spectra were acquired from full elution areas of all detected glycosylated and aglycosylated Im7 species and are representative of n = 2 IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file

    Techniques Used: In Vitro, Mass Spectrometry, Purification, Modification, Produced, Incubation

    30) Product Images from "Dissection of Antibody Specificities Induced by Yellow Fever Vaccination"

    Article Title: Dissection of Antibody Specificities Induced by Yellow Fever Vaccination

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003458

    ELISA and NT analyses of YF post-immunization sera after depletion with YF sE (panels A), YF DI+II (panels B), YF DIII (panels C) and YF prM (panels D). Left panels: Percent reactivity in post-depletion sera, determined by ELISA with the depletion antigen. Middle panels: Percent reactivity in post-depletion sera, determined by ELISA with the virion. Right panels: Percent reactivity in post-depletion sera, determined by NT. Results are expressed as percent of the ELISA IgG units or NT titers of mock-depleted sera (control). The numbers above the columns in the middle and right panels indicate the percentage of reactivities remaining after depletion. Asterisks indicate the significance of difference in antibody reactivities between depleted and control sera (t-test). The error bars represent standard error of the means calculated from the results of at least three independent assays. Identification numbers of vaccinees are indicated under the panels (compare with Table 3 and Figure 4 ).
    Figure Legend Snippet: ELISA and NT analyses of YF post-immunization sera after depletion with YF sE (panels A), YF DI+II (panels B), YF DIII (panels C) and YF prM (panels D). Left panels: Percent reactivity in post-depletion sera, determined by ELISA with the depletion antigen. Middle panels: Percent reactivity in post-depletion sera, determined by ELISA with the virion. Right panels: Percent reactivity in post-depletion sera, determined by NT. Results are expressed as percent of the ELISA IgG units or NT titers of mock-depleted sera (control). The numbers above the columns in the middle and right panels indicate the percentage of reactivities remaining after depletion. Asterisks indicate the significance of difference in antibody reactivities between depleted and control sera (t-test). The error bars represent standard error of the means calculated from the results of at least three independent assays. Identification numbers of vaccinees are indicated under the panels (compare with Table 3 and Figure 4 ).

    Techniques Used: Enzyme-linked Immunosorbent Assay, T-Test

    31) Product Images from "m6A-dependent regulation of messenger RNA stability"

    Article Title: m6A-dependent regulation of messenger RNA stability

    Journal: Nature

    doi: 10.1038/nature12730

    YTHDF2 destabilizes its cognate mRNAs a–d , Cumulative distribution of mRNA input ( a ), ribosome-protected fragments ( b ), and mRNA lifetime log 2 fold changes (Δ, c ) between siYTHDF2 (YTHDF2 knockdown) and siControl (knockdown control) for non-targets (grey), PAR-CLIP targets (blue), and PAR CLIP-RIP common targets (red). The mRNA lifetime log 2 fold changes were further grouped and analyzed based on the number of CLIP sites on each transcript ( d ). The increased binding of YTHDF2 on its target transcript correlates with reduced mRNA lifetime. P values were calculated using two-sided Mann-Whitney or Kruskal-Wallis test (rank-sum test for the comparison of two or multiple samples, respectively). Detailed statistics were presented in Extended Data Fig. 3c . e , Western-blotting of flag-tagged YTHDF2 on each fraction of 10–50% sucrose gradient showing that YTHDF2 does not associate with ribosome. The fractions were grouped to non-ribosome mRNPs, 40–80S, and polysome. f , Quantification of the m 6 A/A ratio of the total mRNA, non-ribosome portion, 40–80S, and polysome by LC-MS/MS. Noticeable increases of the m 6 A/A ratio of the total mRNA, mRNA from 40–80S, and mRNA from polysome were observed in the siYTHDF2 sample compared to control after 48 h. A reduced m 6 A/A ratio of mRNA isolated from the non-ribosome portion was observed in the same experiment. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d, for poly(A)-tailed total mRNA input, n = 10 (five biological replicates × two technical replicates), and for the rest, n = 4 (two biological replicates × two technical replicates).
    Figure Legend Snippet: YTHDF2 destabilizes its cognate mRNAs a–d , Cumulative distribution of mRNA input ( a ), ribosome-protected fragments ( b ), and mRNA lifetime log 2 fold changes (Δ, c ) between siYTHDF2 (YTHDF2 knockdown) and siControl (knockdown control) for non-targets (grey), PAR-CLIP targets (blue), and PAR CLIP-RIP common targets (red). The mRNA lifetime log 2 fold changes were further grouped and analyzed based on the number of CLIP sites on each transcript ( d ). The increased binding of YTHDF2 on its target transcript correlates with reduced mRNA lifetime. P values were calculated using two-sided Mann-Whitney or Kruskal-Wallis test (rank-sum test for the comparison of two or multiple samples, respectively). Detailed statistics were presented in Extended Data Fig. 3c . e , Western-blotting of flag-tagged YTHDF2 on each fraction of 10–50% sucrose gradient showing that YTHDF2 does not associate with ribosome. The fractions were grouped to non-ribosome mRNPs, 40–80S, and polysome. f , Quantification of the m 6 A/A ratio of the total mRNA, non-ribosome portion, 40–80S, and polysome by LC-MS/MS. Noticeable increases of the m 6 A/A ratio of the total mRNA, mRNA from 40–80S, and mRNA from polysome were observed in the siYTHDF2 sample compared to control after 48 h. A reduced m 6 A/A ratio of mRNA isolated from the non-ribosome portion was observed in the same experiment. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d, for poly(A)-tailed total mRNA input, n = 10 (five biological replicates × two technical replicates), and for the rest, n = 4 (two biological replicates × two technical replicates).

    Techniques Used: Cross-linking Immunoprecipitation, Binding Assay, MANN-WHITNEY, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation

    YTHDF2 selectively binds m 6 A-containing RNA a , Illustration of m 6 A methylatransferase, demethylase, and binding proteins. RRACH is the extended m 6 A consensus motif, where R is G or A and H is not G. b , LC-MS/MS showing m 6 A enrichment in GST-YTHDF2-bound mRNA while depleted in the flow-through portion. Error bars, mean ±s.t.d., n = 2, technical replicates. c , Overlap of peaks identified through YTHDF2-based PAR-CLIP and the m 6 A-seq peaks in the same cell line. d , Binding motif identified by MEME with PAR-CLIP peaks ( p = 3.0 e−46, 381 sites were found under this motif out of top 1000 scored peaks). e , Pie chart depicting the region distribution of YTHDF2-binding sites identified by PAR-CLIP, TTS (200 bp window from the transcription starting site), stop codon (400 bp window centered on stop codon).
    Figure Legend Snippet: YTHDF2 selectively binds m 6 A-containing RNA a , Illustration of m 6 A methylatransferase, demethylase, and binding proteins. RRACH is the extended m 6 A consensus motif, where R is G or A and H is not G. b , LC-MS/MS showing m 6 A enrichment in GST-YTHDF2-bound mRNA while depleted in the flow-through portion. Error bars, mean ±s.t.d., n = 2, technical replicates. c , Overlap of peaks identified through YTHDF2-based PAR-CLIP and the m 6 A-seq peaks in the same cell line. d , Binding motif identified by MEME with PAR-CLIP peaks ( p = 3.0 e−46, 381 sites were found under this motif out of top 1000 scored peaks). e , Pie chart depicting the region distribution of YTHDF2-binding sites identified by PAR-CLIP, TTS (200 bp window from the transcription starting site), stop codon (400 bp window centered on stop codon).

    Techniques Used: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Flow Cytometry, Cross-linking Immunoprecipitation

    YTHDF2 affects SON mRNA localization in processing body (P-body) a , Schematic of the domain architecture (aa stands for amino acids) of YTHDF2, N-terminal of YTHDF2 (N-YTHDF2, aa 1–389, blue) and C-terminal of YTHDF2 (C-YTHDF2, aa 390-end, red). b , Over-expression of full-length YTHDF2 led to reduced levels of m 6 A after 24 h, while over-expression of N-YTHDF2 or C-YTHDF2 increased the m 6 A/A ratio of the total mRNA. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 4 (two biological replicates × two technical replicates). c–e , Fluorescence in situ hybridization of SON mRNA and fluorescence immunostaining of DCP1a (P-body marker), flag-tagged YTHDF2 ( c ), flag-tagged C-YTHDF2, ( d ) and flag-tagged N-YTHDF2 ( e ). Full-length YTHDF2 and C-YTHDF2 co-localize with SON mRNA (bearing m 6 A) while the full-length YTHDF2 significantly increases the P-body localization of SON mRNA compared to N-YTHDF2 and C-YTDF2. The numbers shown above figures are Pearson correlation coefficients of each channel pair with the scale of the magnified region (white frame) set as 2 µm × 2 µm. f , Tethering N-YTHDF2-λ to a mRNA reporter F-luc-5BoxB led to a ~40% reduction of the reporter mRNA level compared to tethering N-YTHDF2 or λ alone (green) and controls without BoxB (F-luc, yellow). P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 6 (F-luc-5BoxB) or 3 (F-luc). g , A proposed model of m 6 A-dependent mRNA degradation mediated through YTHDF2. The three states of mRNAs in cytoplasm are defined by their engagement with ribosome using the sedimentation coefficient range in sucrose gradient: > 80S for actively translating polysome; 40–80S for translatable pool; 20–35S for non-ribosome mRNPs.
    Figure Legend Snippet: YTHDF2 affects SON mRNA localization in processing body (P-body) a , Schematic of the domain architecture (aa stands for amino acids) of YTHDF2, N-terminal of YTHDF2 (N-YTHDF2, aa 1–389, blue) and C-terminal of YTHDF2 (C-YTHDF2, aa 390-end, red). b , Over-expression of full-length YTHDF2 led to reduced levels of m 6 A after 24 h, while over-expression of N-YTHDF2 or C-YTHDF2 increased the m 6 A/A ratio of the total mRNA. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 4 (two biological replicates × two technical replicates). c–e , Fluorescence in situ hybridization of SON mRNA and fluorescence immunostaining of DCP1a (P-body marker), flag-tagged YTHDF2 ( c ), flag-tagged C-YTHDF2, ( d ) and flag-tagged N-YTHDF2 ( e ). Full-length YTHDF2 and C-YTHDF2 co-localize with SON mRNA (bearing m 6 A) while the full-length YTHDF2 significantly increases the P-body localization of SON mRNA compared to N-YTHDF2 and C-YTDF2. The numbers shown above figures are Pearson correlation coefficients of each channel pair with the scale of the magnified region (white frame) set as 2 µm × 2 µm. f , Tethering N-YTHDF2-λ to a mRNA reporter F-luc-5BoxB led to a ~40% reduction of the reporter mRNA level compared to tethering N-YTHDF2 or λ alone (green) and controls without BoxB (F-luc, yellow). P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 6 (F-luc-5BoxB) or 3 (F-luc). g , A proposed model of m 6 A-dependent mRNA degradation mediated through YTHDF2. The three states of mRNAs in cytoplasm are defined by their engagement with ribosome using the sedimentation coefficient range in sucrose gradient: > 80S for actively translating polysome; 40–80S for translatable pool; 20–35S for non-ribosome mRNPs.

    Techniques Used: Over Expression, Fluorescence, In Situ Hybridization, Immunostaining, Marker, Sedimentation

    32) Product Images from "Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot"

    Article Title: Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0233-x

    Purification of minichromosomes (MiniCs) from highly synchronous meiosis. a Structure of MiniCs. Recombination hotspot ( M26 ) and basal control ( BC ) MiniCs contain different alleles of the ade6 gene, a fission yeast origin of replication ( ARS ) and copies of the LacO DNA site for affinity purification. b Efficiency and synchrony of induced meiosis. Plots show the frequencies of cells undergoing the first meiotic division ( MI , 2 nuclei) and having completed the second meiotic division ( MII , 3–4 nuclei) in strains harboring the indicated MiniCs. c The indicated samples of chromatin from steps of purification were deproteinized, and their DNAs were analyzed by agarose gel electrophoresis ( WCE , whole-cell extract). d MiniC copy number and degree of enrichment; note log scale. The abundance of ade6 DNA in the chromosome ( Chr ) or in the MiniC (with chromosomal ade6 deleted) was determined by qPCR, relative to the act1 locus, and those values were normalized relative to single-copy ade6 in the chromosome. Affinity purifications ( AP ) employed LacI-6xHis-prA; mock AP samples were processed identically, but lacked LacI-6xHis-prA. In this figure and others, plots with error bars are mean ± SD from three or more biological replicates
    Figure Legend Snippet: Purification of minichromosomes (MiniCs) from highly synchronous meiosis. a Structure of MiniCs. Recombination hotspot ( M26 ) and basal control ( BC ) MiniCs contain different alleles of the ade6 gene, a fission yeast origin of replication ( ARS ) and copies of the LacO DNA site for affinity purification. b Efficiency and synchrony of induced meiosis. Plots show the frequencies of cells undergoing the first meiotic division ( MI , 2 nuclei) and having completed the second meiotic division ( MII , 3–4 nuclei) in strains harboring the indicated MiniCs. c The indicated samples of chromatin from steps of purification were deproteinized, and their DNAs were analyzed by agarose gel electrophoresis ( WCE , whole-cell extract). d MiniC copy number and degree of enrichment; note log scale. The abundance of ade6 DNA in the chromosome ( Chr ) or in the MiniC (with chromosomal ade6 deleted) was determined by qPCR, relative to the act1 locus, and those values were normalized relative to single-copy ade6 in the chromosome. Affinity purifications ( AP ) employed LacI-6xHis-prA; mock AP samples were processed identically, but lacked LacI-6xHis-prA. In this figure and others, plots with error bars are mean ± SD from three or more biological replicates

    Techniques Used: Purification, Affinity Purification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    33) Product Images from "Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of Borrelia burgdorferi"

    Article Title: Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of Borrelia burgdorferi

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2012/349657

    CRASP-4 binds distinct complement proteins. Binding of equimolar amounts of CFH, CFHR1, CFHR2, and CFHR5 (33 μ M) to immobilized CRASP-4 (5 μ g/mL) was analyzed by ELISA. Bound CFH or CFHR proteins were detected with either goat CFH polyclonal antiserum or mouse CFHR1 monoclonal antiserum (JHD 7.10), which reacts with all three CFHRs. Data represent the means and standard errors from three separate experiments.
    Figure Legend Snippet: CRASP-4 binds distinct complement proteins. Binding of equimolar amounts of CFH, CFHR1, CFHR2, and CFHR5 (33 μ M) to immobilized CRASP-4 (5 μ g/mL) was analyzed by ELISA. Bound CFH or CFHR proteins were detected with either goat CFH polyclonal antiserum or mouse CFHR1 monoclonal antiserum (JHD 7.10), which reacts with all three CFHRs. Data represent the means and standard errors from three separate experiments.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    Characterization of B. garinii G1 producing CRASP-4. (a) B. garinii G1 and transformed strains G1/pKFSS1 and G1/pCRASP-4 were characterized by PCR amplification using flaB- , aadA- , and erpC -specific primers, as listed in Table 1 . (b) Synthesis of CRASP-4 by transformed G1 was assessed using ligand affinity blotting. Whole cell lysates (15 μ g each) of G1, G1/pKFSS1 and G1/pCRASP-4 were separated by SDS-PAGE, and transferred to nitrocellulose. After incubation with NHS, binding of CFH to CRASP-4 was identified using a polyclonal antiserum. A monoclonal antibody, L41 1C11, specific for the flagellin protein FlaB, was applied to show equal loading of borrelial lysates. (c) Surface localization of CRASP-4 in transformed G1 cells. Spirochetes were incubated with or without proteinase K or trypsin, respectively, then lysed by sonication, and total proteins were separated by SDS-PAGE. CRASP-4 was identified by ligand affinity analysis as described above. Flagellin (FlaB) was detected with MAb L41 1C11 (dilution 1/1000) by Western blotting. (d) Demonstration of surface expression of CRASP-4 by transformed B. garinii G1, by indirect immunofluoresecence microscopy of intact borrelial cells. Spirochetes were incubated with rabbit polyclonal anti-ErpA/ErpC antiserum before fixation. Periplasmic FlaB, used as control, was detected by mAb L41 1C11 using fixed and unfixed cells. For counterstaining, the DNA-binding dye DAPI was used to identify all bacteria. Slides were visualized at a magnification of ×1,000 using an Olympus CX40 fluorescence microscope mounted with a DS-5Mc charge-coupled device camera (Nikon).
    Figure Legend Snippet: Characterization of B. garinii G1 producing CRASP-4. (a) B. garinii G1 and transformed strains G1/pKFSS1 and G1/pCRASP-4 were characterized by PCR amplification using flaB- , aadA- , and erpC -specific primers, as listed in Table 1 . (b) Synthesis of CRASP-4 by transformed G1 was assessed using ligand affinity blotting. Whole cell lysates (15 μ g each) of G1, G1/pKFSS1 and G1/pCRASP-4 were separated by SDS-PAGE, and transferred to nitrocellulose. After incubation with NHS, binding of CFH to CRASP-4 was identified using a polyclonal antiserum. A monoclonal antibody, L41 1C11, specific for the flagellin protein FlaB, was applied to show equal loading of borrelial lysates. (c) Surface localization of CRASP-4 in transformed G1 cells. Spirochetes were incubated with or without proteinase K or trypsin, respectively, then lysed by sonication, and total proteins were separated by SDS-PAGE. CRASP-4 was identified by ligand affinity analysis as described above. Flagellin (FlaB) was detected with MAb L41 1C11 (dilution 1/1000) by Western blotting. (d) Demonstration of surface expression of CRASP-4 by transformed B. garinii G1, by indirect immunofluoresecence microscopy of intact borrelial cells. Spirochetes were incubated with rabbit polyclonal anti-ErpA/ErpC antiserum before fixation. Periplasmic FlaB, used as control, was detected by mAb L41 1C11 using fixed and unfixed cells. For counterstaining, the DNA-binding dye DAPI was used to identify all bacteria. Slides were visualized at a magnification of ×1,000 using an Olympus CX40 fluorescence microscope mounted with a DS-5Mc charge-coupled device camera (Nikon).

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Amplification, SDS Page, Incubation, Binding Assay, Sonication, Western Blot, Expressing, Microscopy, Fluorescence

    Identification of serum proteins that bind to recombinant CRASP-4. Recombinant, polyhistidine-tagged CRASP-4 was immobilized onto magnetic beads and incubated with NHS. Uncoated beads were also treated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100mM glycine-HCl (pH 2.0) and the eluate fractions were separated by SDS-PAGE under nonreducing conditions. (a) Silver stain of a gel loaded with purified polyhistidine-tagged CRASP-4 (1 μ g), eluate fraction of the uncoated beads, and the final wash and eluate fraction of CRASP-4-coated beads. (b) Western blot analysis of the eluate fraction of CRASP-4-coated beads using a polyclonal anti-CFH or a polyclonal anti-CFHR1 antiserum. Mobilities of molecular mass standards are indicated to the left.
    Figure Legend Snippet: Identification of serum proteins that bind to recombinant CRASP-4. Recombinant, polyhistidine-tagged CRASP-4 was immobilized onto magnetic beads and incubated with NHS. Uncoated beads were also treated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100mM glycine-HCl (pH 2.0) and the eluate fractions were separated by SDS-PAGE under nonreducing conditions. (a) Silver stain of a gel loaded with purified polyhistidine-tagged CRASP-4 (1 μ g), eluate fraction of the uncoated beads, and the final wash and eluate fraction of CRASP-4-coated beads. (b) Western blot analysis of the eluate fraction of CRASP-4-coated beads using a polyclonal anti-CFH or a polyclonal anti-CFHR1 antiserum. Mobilities of molecular mass standards are indicated to the left.

    Techniques Used: Recombinant, Magnetic Beads, Incubation, Binding Assay, SDS Page, Silver Staining, Purification, Western Blot

    34) Product Images from "m6A-dependent regulation of messenger RNA stability"

    Article Title: m6A-dependent regulation of messenger RNA stability

    Journal: Nature

    doi: 10.1038/nature12730

    YTHDF2 destabilizes its cognate mRNAs a–d , Cumulative distribution of mRNA input ( a ), ribosome-protected fragments ( b ), and mRNA lifetime log 2 fold changes (Δ, c ) between siYTHDF2 (YTHDF2 knockdown) and siControl (knockdown control) for non-targets (grey), PAR-CLIP targets (blue), and PAR CLIP-RIP common targets (red). The mRNA lifetime log 2 fold changes were further grouped and analyzed based on the number of CLIP sites on each transcript ( d ). The increased binding of YTHDF2 on its target transcript correlates with reduced mRNA lifetime. P values were calculated using two-sided Mann-Whitney or Kruskal-Wallis test (rank-sum test for the comparison of two or multiple samples, respectively). Detailed statistics were presented in Extended Data Fig. 3c . e , Western-blotting of flag-tagged YTHDF2 on each fraction of 10–50% sucrose gradient showing that YTHDF2 does not associate with ribosome. The fractions were grouped to non-ribosome mRNPs, 40–80S, and polysome. f , Quantification of the m 6 A/A ratio of the total mRNA, non-ribosome portion, 40–80S, and polysome by LC-MS/MS. Noticeable increases of the m 6 A/A ratio of the total mRNA, mRNA from 40–80S, and mRNA from polysome were observed in the siYTHDF2 sample compared to control after 48 h. A reduced m 6 A/A ratio of mRNA isolated from the non-ribosome portion was observed in the same experiment. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d, for poly(A)-tailed total mRNA input, n = 10 (five biological replicates × two technical replicates), and for the rest, n = 4 (two biological replicates × two technical replicates).
    Figure Legend Snippet: YTHDF2 destabilizes its cognate mRNAs a–d , Cumulative distribution of mRNA input ( a ), ribosome-protected fragments ( b ), and mRNA lifetime log 2 fold changes (Δ, c ) between siYTHDF2 (YTHDF2 knockdown) and siControl (knockdown control) for non-targets (grey), PAR-CLIP targets (blue), and PAR CLIP-RIP common targets (red). The mRNA lifetime log 2 fold changes were further grouped and analyzed based on the number of CLIP sites on each transcript ( d ). The increased binding of YTHDF2 on its target transcript correlates with reduced mRNA lifetime. P values were calculated using two-sided Mann-Whitney or Kruskal-Wallis test (rank-sum test for the comparison of two or multiple samples, respectively). Detailed statistics were presented in Extended Data Fig. 3c . e , Western-blotting of flag-tagged YTHDF2 on each fraction of 10–50% sucrose gradient showing that YTHDF2 does not associate with ribosome. The fractions were grouped to non-ribosome mRNPs, 40–80S, and polysome. f , Quantification of the m 6 A/A ratio of the total mRNA, non-ribosome portion, 40–80S, and polysome by LC-MS/MS. Noticeable increases of the m 6 A/A ratio of the total mRNA, mRNA from 40–80S, and mRNA from polysome were observed in the siYTHDF2 sample compared to control after 48 h. A reduced m 6 A/A ratio of mRNA isolated from the non-ribosome portion was observed in the same experiment. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d, for poly(A)-tailed total mRNA input, n = 10 (five biological replicates × two technical replicates), and for the rest, n = 4 (two biological replicates × two technical replicates).

    Techniques Used: Cross-linking Immunoprecipitation, Binding Assay, MANN-WHITNEY, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation

    YTHDF2 selectively binds m 6 A-containing RNA a , Illustration of m 6 A methylatransferase, demethylase, and binding proteins. RRACH is the extended m 6 A consensus motif, where R is G or A and H is not G. b , LC-MS/MS showing m 6 A enrichment in GST-YTHDF2-bound mRNA while depleted in the flow-through portion. Error bars, mean ±s.t.d., n = 2, technical replicates. c , Overlap of peaks identified through YTHDF2-based PAR-CLIP and the m 6 A-seq peaks in the same cell line. d , Binding motif identified by MEME with PAR-CLIP peaks ( p = 3.0 e−46, 381 sites were found under this motif out of top 1000 scored peaks). e , Pie chart depicting the region distribution of YTHDF2-binding sites identified by PAR-CLIP, TTS (200 bp window from the transcription starting site), stop codon (400 bp window centered on stop codon).
    Figure Legend Snippet: YTHDF2 selectively binds m 6 A-containing RNA a , Illustration of m 6 A methylatransferase, demethylase, and binding proteins. RRACH is the extended m 6 A consensus motif, where R is G or A and H is not G. b , LC-MS/MS showing m 6 A enrichment in GST-YTHDF2-bound mRNA while depleted in the flow-through portion. Error bars, mean ±s.t.d., n = 2, technical replicates. c , Overlap of peaks identified through YTHDF2-based PAR-CLIP and the m 6 A-seq peaks in the same cell line. d , Binding motif identified by MEME with PAR-CLIP peaks ( p = 3.0 e−46, 381 sites were found under this motif out of top 1000 scored peaks). e , Pie chart depicting the region distribution of YTHDF2-binding sites identified by PAR-CLIP, TTS (200 bp window from the transcription starting site), stop codon (400 bp window centered on stop codon).

    Techniques Used: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Flow Cytometry, Cross-linking Immunoprecipitation

    YTHDF2 affects SON mRNA localization in processing body (P-body) a , Schematic of the domain architecture (aa stands for amino acids) of YTHDF2, N-terminal of YTHDF2 (N-YTHDF2, aa 1–389, blue) and C-terminal of YTHDF2 (C-YTHDF2, aa 390-end, red). b , Over-expression of full-length YTHDF2 led to reduced levels of m 6 A after 24 h, while over-expression of N-YTHDF2 or C-YTHDF2 increased the m 6 A/A ratio of the total mRNA. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 4 (two biological replicates × two technical replicates). c–e , Fluorescence in situ hybridization of SON mRNA and fluorescence immunostaining of DCP1a (P-body marker), flag-tagged YTHDF2 ( c ), flag-tagged C-YTHDF2, ( d ) and flag-tagged N-YTHDF2 ( e ). Full-length YTHDF2 and C-YTHDF2 co-localize with SON mRNA (bearing m 6 A) while the full-length YTHDF2 significantly increases the P-body localization of SON mRNA compared to N-YTHDF2 and C-YTDF2. The numbers shown above figures are Pearson correlation coefficients of each channel pair with the scale of the magnified region (white frame) set as 2 µm × 2 µm. f , Tethering N-YTHDF2-λ to a mRNA reporter F-luc-5BoxB led to a ~40% reduction of the reporter mRNA level compared to tethering N-YTHDF2 or λ alone (green) and controls without BoxB (F-luc, yellow). P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 6 (F-luc-5BoxB) or 3 (F-luc). g , A proposed model of m 6 A-dependent mRNA degradation mediated through YTHDF2. The three states of mRNAs in cytoplasm are defined by their engagement with ribosome using the sedimentation coefficient range in sucrose gradient: > 80S for actively translating polysome; 40–80S for translatable pool; 20–35S for non-ribosome mRNPs.
    Figure Legend Snippet: YTHDF2 affects SON mRNA localization in processing body (P-body) a , Schematic of the domain architecture (aa stands for amino acids) of YTHDF2, N-terminal of YTHDF2 (N-YTHDF2, aa 1–389, blue) and C-terminal of YTHDF2 (C-YTHDF2, aa 390-end, red). b , Over-expression of full-length YTHDF2 led to reduced levels of m 6 A after 24 h, while over-expression of N-YTHDF2 or C-YTHDF2 increased the m 6 A/A ratio of the total mRNA. P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 4 (two biological replicates × two technical replicates). c–e , Fluorescence in situ hybridization of SON mRNA and fluorescence immunostaining of DCP1a (P-body marker), flag-tagged YTHDF2 ( c ), flag-tagged C-YTHDF2, ( d ) and flag-tagged N-YTHDF2 ( e ). Full-length YTHDF2 and C-YTHDF2 co-localize with SON mRNA (bearing m 6 A) while the full-length YTHDF2 significantly increases the P-body localization of SON mRNA compared to N-YTHDF2 and C-YTDF2. The numbers shown above figures are Pearson correlation coefficients of each channel pair with the scale of the magnified region (white frame) set as 2 µm × 2 µm. f , Tethering N-YTHDF2-λ to a mRNA reporter F-luc-5BoxB led to a ~40% reduction of the reporter mRNA level compared to tethering N-YTHDF2 or λ alone (green) and controls without BoxB (F-luc, yellow). P values were determined using two-sided Student’s t -test for paired samples. Error bars, mean ± s.t.d., n = 6 (F-luc-5BoxB) or 3 (F-luc). g , A proposed model of m 6 A-dependent mRNA degradation mediated through YTHDF2. The three states of mRNAs in cytoplasm are defined by their engagement with ribosome using the sedimentation coefficient range in sucrose gradient: > 80S for actively translating polysome; 40–80S for translatable pool; 20–35S for non-ribosome mRNPs.

    Techniques Used: Over Expression, Fluorescence, In Situ Hybridization, Immunostaining, Marker, Sedimentation

    35) Product Images from "Structural basis for DNA 3′-end processing by human tyrosyl-DNA phosphodiesterase 1"

    Article Title: Structural basis for DNA 3′-end processing by human tyrosyl-DNA phosphodiesterase 1

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02530-z

    Tdp1(Δ148) nucleosidase activity and analysis of the DNA content of Tdp1(Δ148):DNA crystals. Reaction schemes depicting a 32 P-labelling of a 12-mer DNA duplex with PNK, followed by removal of the 3′-nucleoside by Tdp1(Δ148) (star denotes 32 P). b Denaturing PAGE of the products of the reactions shown in A (lane 2) and B (lane 3). The four distinct species in the PNK-treated dissolved crystals (lane 9) are labelled from w to z. Lanes 4–8 contain a 32 P-labelled marker of length 11 nts to 7 nts, respectively. c 3′-nucleoside removal by Tdp1(Δ148), followed by 5′- 32 P-labelling with PNK, which also removes the 3′-phosphate created by Tdp1 cleavage. d Proposed reaction of Tdp1(Δ148) in the crystals, followed by 32 P-labelling of dissolved crystals with PNK
    Figure Legend Snippet: Tdp1(Δ148) nucleosidase activity and analysis of the DNA content of Tdp1(Δ148):DNA crystals. Reaction schemes depicting a 32 P-labelling of a 12-mer DNA duplex with PNK, followed by removal of the 3′-nucleoside by Tdp1(Δ148) (star denotes 32 P). b Denaturing PAGE of the products of the reactions shown in A (lane 2) and B (lane 3). The four distinct species in the PNK-treated dissolved crystals (lane 9) are labelled from w to z. Lanes 4–8 contain a 32 P-labelled marker of length 11 nts to 7 nts, respectively. c 3′-nucleoside removal by Tdp1(Δ148), followed by 5′- 32 P-labelling with PNK, which also removes the 3′-phosphate created by Tdp1 cleavage. d Proposed reaction of Tdp1(Δ148) in the crystals, followed by 32 P-labelling of dissolved crystals with PNK

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Marker

    Crystal structures of Tdp1(Δ148)–DNA. a Quaternary transition-state complex of Tdp1(Δ148), ssDNA (orange), a Top1-derived peptide (pink) and vanadate (PDB ID: 1NOP). b Tdp1(Δ148) in complex with the −2G DNA duplex (PDB ID: 5NW9). c Tdp1(Δ148) in complex with the −2T DNA duplex (PDB ID: 5NWA). For a – c , Tdp1(Δ148) is displayed as an electrostatic surface (blue indicates positive charge and red negative charge). The sequence of the DNA in each structure that is clearly defined by the electron density is shown below. The scissile strand is orange and the complementary strand is green. d Tdp1(Δ148) contacts with the complementary strand in the −2T Tdp1(Δ148) DNA complex. Tdp1(Δ148) is shown as an electrostatic surface with underlying structure. The side chains of N528, K527, R361, K231 and R232 are shown as sticks, and interactions with the complementary DNA strand as dotted lines (distances in Ångstrom). e Close-up view of complementary strand interactions with β−turn residues K527 and N528. The structure of Tdp1(Δ148) bound to ssDNA (PDB ID: 1NOP) is superimposed. Loop residues between β15 and β16 are rainbow coloured according to B-factors, with blue indicating the minimum and red the maximum B-factor. f Alignments of Tdp1 sequences from diverse species, with K527 (blue star) marked and the secondary structure elements shown above the alignment
    Figure Legend Snippet: Crystal structures of Tdp1(Δ148)–DNA. a Quaternary transition-state complex of Tdp1(Δ148), ssDNA (orange), a Top1-derived peptide (pink) and vanadate (PDB ID: 1NOP). b Tdp1(Δ148) in complex with the −2G DNA duplex (PDB ID: 5NW9). c Tdp1(Δ148) in complex with the −2T DNA duplex (PDB ID: 5NWA). For a – c , Tdp1(Δ148) is displayed as an electrostatic surface (blue indicates positive charge and red negative charge). The sequence of the DNA in each structure that is clearly defined by the electron density is shown below. The scissile strand is orange and the complementary strand is green. d Tdp1(Δ148) contacts with the complementary strand in the −2T Tdp1(Δ148) DNA complex. Tdp1(Δ148) is shown as an electrostatic surface with underlying structure. The side chains of N528, K527, R361, K231 and R232 are shown as sticks, and interactions with the complementary DNA strand as dotted lines (distances in Ångstrom). e Close-up view of complementary strand interactions with β−turn residues K527 and N528. The structure of Tdp1(Δ148) bound to ssDNA (PDB ID: 1NOP) is superimposed. Loop residues between β15 and β16 are rainbow coloured according to B-factors, with blue indicating the minimum and red the maximum B-factor. f Alignments of Tdp1 sequences from diverse species, with K527 (blue star) marked and the secondary structure elements shown above the alignment

    Techniques Used: Derivative Assay, Sequencing

    Tdp1 is a DNA 3′-end processing enzyme. Schematic representation of Tdp1 activity (represented by scissors) on biologically and medically relevant substrates. All Tdp1 reactions result in DNA with a 3′-phosphorylated end. a Hydrolysis of the phosphotyrosyl linkage between a proteolytic topoisomerase 1 fragment and the 3′-end of the DNA at a nick. b Removal of glycolate from 3′ overhangs with a phosphoglycolate (PG) adduct. c Tdp1 nucleosidase activity on unmodified DNA with a 3′-hydroxyl end. d Removal of chain-terminating nucleoside analogues (CTNAs), such as AZT (zidovudine) from a recessed 3′ end
    Figure Legend Snippet: Tdp1 is a DNA 3′-end processing enzyme. Schematic representation of Tdp1 activity (represented by scissors) on biologically and medically relevant substrates. All Tdp1 reactions result in DNA with a 3′-phosphorylated end. a Hydrolysis of the phosphotyrosyl linkage between a proteolytic topoisomerase 1 fragment and the 3′-end of the DNA at a nick. b Removal of glycolate from 3′ overhangs with a phosphoglycolate (PG) adduct. c Tdp1 nucleosidase activity on unmodified DNA with a 3′-hydroxyl end. d Removal of chain-terminating nucleoside analogues (CTNAs), such as AZT (zidovudine) from a recessed 3′ end

    Techniques Used: Activity Assay

    PD-XLMS method for site-specific protein–DNA cross-linking with mass spectrometry. Tdp1(Δ148) protein (blue, H263A inactive mutant) is cross-linked to DNA (black line) containing 5-Iodouracil (5IdU) and a 3′-Biotin-TEG (depicted as B in a red circle). Small blue circles represent Tdp1 amino acids (after trypsin digestion) and the red star indicates the site of the cross-link
    Figure Legend Snippet: PD-XLMS method for site-specific protein–DNA cross-linking with mass spectrometry. Tdp1(Δ148) protein (blue, H263A inactive mutant) is cross-linked to DNA (black line) containing 5-Iodouracil (5IdU) and a 3′-Biotin-TEG (depicted as B in a red circle). Small blue circles represent Tdp1 amino acids (after trypsin digestion) and the red star indicates the site of the cross-link

    Techniques Used: Mass Spectrometry, Mutagenesis

    Tdp1(Δ148) cross-links via F259 to −2 and −3 modified nucleobases. a Schematic of the single-stranded DNA 20-mers used in cross-linking experiments. Modified oligonucleotides have 5-Iodouracil (5IdU) at the −2 or −3 position. b Eight per cent SDS-PAGE of DNA oligonucleotides, containing a 32 P-label (star), cross-linked to catalytically inactive Tdp1(Δ148) H263A, visualised by phosphorimaging (upper) and SimplyBlue TM staining (lower). c LC elution chromatograms (at 260 nm) of samples cross-linked to the −3 5IdU (blue), −2 5IdU (red) and unmodified control (black) oligonucleotides. d Negative-mode mass spectra showing the charge-state distribution of cross-linked Tdp1(Δ148) peptide–DNA heteroconjugates (cross-linked to the −3 5IdU (blue), −2 5IdU (red) and unmodified control (black) oligonucleotides) eluting from the LC between 15 and 17 min after injection. e Positive-mode mass spectra of −3 5IdU (blue), −2 5IdU (red) and control (black) cross-linked, trypsin and nuclease digested samples. Unique [M+2H] 2+ ions (marked by arrows) are observed at m/z 740.33269 with both 5IdU cross-linked samples. f Collision-induced dissociation fragmentation (CID) mass spectrum of the [M+2H] 2+ ion at m/z 740.33 at 20 V (for the −2 cross-link sample) and the sequence of the cross-linked Tdp1 peptide. The position of the DNA cross-link (F, shaded in red) is determined by the presence of modified fragment ions (annotated in red) containing a single deoxyuracil monophosphate (dUMP, annotated with ##) or a uracil base (annotated by #), which arises from fragmentation of the glycosidic ribose–base bond during CID. Identified peptide b and y fragment ions are annotated in black
    Figure Legend Snippet: Tdp1(Δ148) cross-links via F259 to −2 and −3 modified nucleobases. a Schematic of the single-stranded DNA 20-mers used in cross-linking experiments. Modified oligonucleotides have 5-Iodouracil (5IdU) at the −2 or −3 position. b Eight per cent SDS-PAGE of DNA oligonucleotides, containing a 32 P-label (star), cross-linked to catalytically inactive Tdp1(Δ148) H263A, visualised by phosphorimaging (upper) and SimplyBlue TM staining (lower). c LC elution chromatograms (at 260 nm) of samples cross-linked to the −3 5IdU (blue), −2 5IdU (red) and unmodified control (black) oligonucleotides. d Negative-mode mass spectra showing the charge-state distribution of cross-linked Tdp1(Δ148) peptide–DNA heteroconjugates (cross-linked to the −3 5IdU (blue), −2 5IdU (red) and unmodified control (black) oligonucleotides) eluting from the LC between 15 and 17 min after injection. e Positive-mode mass spectra of −3 5IdU (blue), −2 5IdU (red) and control (black) cross-linked, trypsin and nuclease digested samples. Unique [M+2H] 2+ ions (marked by arrows) are observed at m/z 740.33269 with both 5IdU cross-linked samples. f Collision-induced dissociation fragmentation (CID) mass spectrum of the [M+2H] 2+ ion at m/z 740.33 at 20 V (for the −2 cross-link sample) and the sequence of the cross-linked Tdp1 peptide. The position of the DNA cross-link (F, shaded in red) is determined by the presence of modified fragment ions (annotated in red) containing a single deoxyuracil monophosphate (dUMP, annotated with ##) or a uracil base (annotated by #), which arises from fragmentation of the glycosidic ribose–base bond during CID. Identified peptide b and y fragment ions are annotated in black

    Techniques Used: Modification, SDS Page, Staining, Injection, Sequencing

    36) Product Images from "Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2"

    Article Title: Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16966-3

    HULC interacts with the glycolytic enzyme LDHA. a Validation of the interaction between LDHA and HULC. Immunoblots of LDHA in the cell lysates and immunoprecipitates of LDHA are shown in the left panel. Agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the right panel. LincRNA-p21 was examined as the RNA control. b The cellular localizations of HULC and LDHA were analyzed by combining RNA-FISH and immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Biotinylated HULC was incubated with HepG2 cell lysate and then isolated by streptavidin-conjugated beads. LDHA and LDHB in the cell lysate and RNA pull-down were examined by western blotting. Biotinylated antisense HULC was used as the control. d His-tagged rLDHA or rLDHB was incubated with Dynabeads® His-tag isolation magnetic beads, respectively. Next, in vitro transcribed HULC or antisense HULC was incubated with the beads. Then, the RNA-protein complexes were isolated, and the levels of HULC were examined using qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P
    Figure Legend Snippet: HULC interacts with the glycolytic enzyme LDHA. a Validation of the interaction between LDHA and HULC. Immunoblots of LDHA in the cell lysates and immunoprecipitates of LDHA are shown in the left panel. Agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the right panel. LincRNA-p21 was examined as the RNA control. b The cellular localizations of HULC and LDHA were analyzed by combining RNA-FISH and immunofluorescence. Cell nuclei were stained with DAPI, and the scale bar was 20 μm. c Biotinylated HULC was incubated with HepG2 cell lysate and then isolated by streptavidin-conjugated beads. LDHA and LDHB in the cell lysate and RNA pull-down were examined by western blotting. Biotinylated antisense HULC was used as the control. d His-tagged rLDHA or rLDHB was incubated with Dynabeads® His-tag isolation magnetic beads, respectively. Next, in vitro transcribed HULC or antisense HULC was incubated with the beads. Then, the RNA-protein complexes were isolated, and the levels of HULC were examined using qRT-PCR. Data represent the mean ± s.d. of triplicate independent experiments (*** P

    Techniques Used: Western Blot, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Incubation, Isolation, Magnetic Beads, In Vitro

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    Synthesized:

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    Incubation:

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  • 97
    Thermo Fisher coated magnetic dynabeads
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    Thermo Fisher his tag dynabeadstm magnetic beads
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    Thermo Fisher magnetic beads
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    Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.

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    Figure Lengend Snippet: Nanobody-coated beads agglutinate C. jejuni KC40 cells. The His-tagged nanobodies were coupled to magnetic dynabeads, leading to multimerization. A Nb84 coupled to dynabeads causes agglutination of KC40 cells. B As a negative control, dynabeads coated with anti- E. coli nanobodies were mixed with KC40 cells. No agglutination was observed in this case. C The C. jejuni KC40 bacteria and D the beads coated with Nb84. The results were observed by phase contrast microscopy, using a ×100 oil immersion objective.

    Article Snippet: Multimerization of nanobodies Selected nanobodies were coupled to Co2+ -coated magnetic Dynabeads (Dynabeads® His-Tag Isolation and Pulldown, Thermo Fisher Scientific) to make them multivalent, using the buffers described in the manual.

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