pngase f  (New England Biolabs)


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    Name:
    PNGase F Recombinant
    Description:
    PNGase F Recombinant 75 000 units
    Catalog Number:
    P0708L
    Price:
    641
    Category:
    Glycosidases
    Size:
    75 000 units
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    New England Biolabs pngase f
    PNGase F Recombinant
    PNGase F Recombinant 75 000 units
    https://www.bioz.com/result/pngase f/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pngase f - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Glycosylation of Twisted Gastrulation is Required for BMP Binding and Activity during Craniofacial Development"

    Article Title: Glycosylation of Twisted Gastrulation is Required for BMP Binding and Activity during Craniofacial Development

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2011.00059

    TWSG1 glycosylation varies depending on host source . mTWSG1 made in murine myeloma cells is markedly glycosylated as indicated by the increase in mobility with PNGase F treatment. Xenopus Tsg made in insect cells is also glycosylated but shows a smaller mobility shift. Murine TWSG1 made in E. coli is not glycosylated and shows no shift in mobility after treatment with PNGase F.
    Figure Legend Snippet: TWSG1 glycosylation varies depending on host source . mTWSG1 made in murine myeloma cells is markedly glycosylated as indicated by the increase in mobility with PNGase F treatment. Xenopus Tsg made in insect cells is also glycosylated but shows a smaller mobility shift. Murine TWSG1 made in E. coli is not glycosylated and shows no shift in mobility after treatment with PNGase F.

    Techniques Used: Mobility Shift

    2) Product Images from "An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells"

    Article Title: An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097574

    Purification of the gHA1 from insect cells. (A) SDS-PAGE analysis of AIV HA protein expressed in a baculovirus/insect cell system. His-tagged recombinant hemagglutinin protein (gHA1) was purified using Ni-NTA affinity chromatography and gel filtration. Whole supernatant of insect cell (TriEx Sf9) culture was first loaded onto a Ni-NTA affinity chromatography column (Lane 1). Lanes 2 and 3 are the flow-through and washing eluants through the Ni-NTA affinity column, respectively. Lanes 4 to 6 are collected fractions by imidazole elution. Lane 7 is purified gHA1 after gel filtration chromatography (indicated with an arrow). (B) Cleavage of glycans from the purified gHA1. The purified gHA1 (3 µg) was incubated with (+) or without (–) PNGase F, and the reaction mixture was resolved with 12% SDS-PAGE (left panel). HA was probed with anti-HA polyclonal antibody (right panel). The migration of PNGase F treated (+) and untreated (–) recombinant HA1 is indicated with arrows.
    Figure Legend Snippet: Purification of the gHA1 from insect cells. (A) SDS-PAGE analysis of AIV HA protein expressed in a baculovirus/insect cell system. His-tagged recombinant hemagglutinin protein (gHA1) was purified using Ni-NTA affinity chromatography and gel filtration. Whole supernatant of insect cell (TriEx Sf9) culture was first loaded onto a Ni-NTA affinity chromatography column (Lane 1). Lanes 2 and 3 are the flow-through and washing eluants through the Ni-NTA affinity column, respectively. Lanes 4 to 6 are collected fractions by imidazole elution. Lane 7 is purified gHA1 after gel filtration chromatography (indicated with an arrow). (B) Cleavage of glycans from the purified gHA1. The purified gHA1 (3 µg) was incubated with (+) or without (–) PNGase F, and the reaction mixture was resolved with 12% SDS-PAGE (left panel). HA was probed with anti-HA polyclonal antibody (right panel). The migration of PNGase F treated (+) and untreated (–) recombinant HA1 is indicated with arrows.

    Techniques Used: Purification, SDS Page, Recombinant, Affinity Chromatography, Filtration, Affinity Column, Flow Cytometry, Chromatography, Incubation, Migration

    3) Product Images from "Identification and Characterization of TEX101 in Bovine Epididymal Spermatozoa"

    Article Title: Identification and Characterization of TEX101 in Bovine Epididymal Spermatozoa

    Journal: Biochemistry Research International

    doi: 10.1155/2014/573293

    (a) Immunoblots of cauda sperm plasma membrane fraction treated with N-glycanase analyzed by reducing SDS-PAGE on 15% gels and immunostained with anti-TEX101. Lane 1 represents the untreated TEX101 polypeptide, and lane 2 displays N-glycanase treated TEX101 polypeptide. Note the reduction in molecular weight (~17 kDa) of the deglycosylated sample. Each lane contains 15 μ g protein. (b) Western blot analysis of TEX101 polypeptide treated with PIPLC and immunostained with anti-TEX101. Lanes 1 and 2 display the untreated plasma membranes (control) of pellet and supernatant fractions, respectively. TEX101 is present in the pellet fraction (lane 1). Lanes 3 and 4 exhibit the pellet and the supernatant fractions of PIPLC treated plasma membranes, respectively. Note that there is a complete release of TEX101 polypeptide in the supernatant fraction (lane 4) of PIPLC treated plasma membranes. The amount of plasma membrane proteins employed in the control and PIPLC treated experiments was 15 μ g.
    Figure Legend Snippet: (a) Immunoblots of cauda sperm plasma membrane fraction treated with N-glycanase analyzed by reducing SDS-PAGE on 15% gels and immunostained with anti-TEX101. Lane 1 represents the untreated TEX101 polypeptide, and lane 2 displays N-glycanase treated TEX101 polypeptide. Note the reduction in molecular weight (~17 kDa) of the deglycosylated sample. Each lane contains 15 μ g protein. (b) Western blot analysis of TEX101 polypeptide treated with PIPLC and immunostained with anti-TEX101. Lanes 1 and 2 display the untreated plasma membranes (control) of pellet and supernatant fractions, respectively. TEX101 is present in the pellet fraction (lane 1). Lanes 3 and 4 exhibit the pellet and the supernatant fractions of PIPLC treated plasma membranes, respectively. Note that there is a complete release of TEX101 polypeptide in the supernatant fraction (lane 4) of PIPLC treated plasma membranes. The amount of plasma membrane proteins employed in the control and PIPLC treated experiments was 15 μ g.

    Techniques Used: Western Blot, SDS Page, Molecular Weight

    4) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.478578

    N -Glycosylation altered the molecular mass and biochemical properties of LILRA3. A , Western blotting of PNGase F-treated ( P +) and PNGase F-untreated ( P −) rLILRA3 from 293T cells and  P. pastoris  using anti-LILRA3 mAb showed a substantial reduction in molecular mass of both eukaryotic cell-produced recombinant proteins following deglycosylation. Recombinant LILRA3 produced in  E. coli  served as a control for non-glycosylated protein. Non-PNGase F-treated LILRA3 from native macrophages ( Macs ) of two individual donors ( lanes 1  and  2 ) is shown as positive references to optimally glycosylated protein.  B , silver staining of two-dimensional gel of non-deglycosylated purified rLILRA3 from 293T cells showed a spectrum of isoelectric focusing with pI ranging from 6 to 9 ( upper panel ), but upon deglycosylation using PNGase F, it was reduced to a single focus with a pI of 7 ( lower panel ).
    Figure Legend Snippet: N -Glycosylation altered the molecular mass and biochemical properties of LILRA3. A , Western blotting of PNGase F-treated ( P +) and PNGase F-untreated ( P −) rLILRA3 from 293T cells and P. pastoris using anti-LILRA3 mAb showed a substantial reduction in molecular mass of both eukaryotic cell-produced recombinant proteins following deglycosylation. Recombinant LILRA3 produced in E. coli served as a control for non-glycosylated protein. Non-PNGase F-treated LILRA3 from native macrophages ( Macs ) of two individual donors ( lanes 1 and 2 ) is shown as positive references to optimally glycosylated protein. B , silver staining of two-dimensional gel of non-deglycosylated purified rLILRA3 from 293T cells showed a spectrum of isoelectric focusing with pI ranging from 6 to 9 ( upper panel ), but upon deglycosylation using PNGase F, it was reduced to a single focus with a pI of 7 ( lower panel ).

    Techniques Used: Western Blot, Produced, Recombinant, Magnetic Cell Separation, Silver Staining, Two-Dimensional Gel Electrophoresis, Purification

    Representative nano-LC-MS/MS of PNGase F-deglycosylated tryptic-digested peptides of mammalian rLILRA3 confirmed four predicted  N -glycosylation sites. A , in-gel peptide digestion of deglycosylated rLILRA3 with Glu-C showed deamidation of asparagine to aspartic acid at Asn 140  ( panel i ), Asn 281  ( panel ii ), and Asn 431  ( panel iii ) indicating  N -linked glycosylation of these sites.  B , digestion with chymotrypsin showed deamidation at Asn 281  ( panel i ) and Asn 341  ( panel ii ).  C , digestion with trypsin detected Asn 281  ( panel i ) and Asn 431  ( panel ii ). It is noteworthy that some sites were detected in peptides digested by more than one enzyme, and none of the enzymes provided full peptide coverage. The predicted Asn 302  was not detected. The sequence of the peptide, the fragmentation pattern, and the detected fragment ions are shown at the  top right  in each panel.  b  ions contain the N-terminal region of the peptide;  y  ions contain the C-terminal region of the peptide. Deamidation of asparagine to aspartic acid is designated as “ N ” with an  underscore .
    Figure Legend Snippet: Representative nano-LC-MS/MS of PNGase F-deglycosylated tryptic-digested peptides of mammalian rLILRA3 confirmed four predicted N -glycosylation sites. A , in-gel peptide digestion of deglycosylated rLILRA3 with Glu-C showed deamidation of asparagine to aspartic acid at Asn 140 ( panel i ), Asn 281 ( panel ii ), and Asn 431 ( panel iii ) indicating N -linked glycosylation of these sites. B , digestion with chymotrypsin showed deamidation at Asn 281 ( panel i ) and Asn 341 ( panel ii ). C , digestion with trypsin detected Asn 281 ( panel i ) and Asn 431 ( panel ii ). It is noteworthy that some sites were detected in peptides digested by more than one enzyme, and none of the enzymes provided full peptide coverage. The predicted Asn 302 was not detected. The sequence of the peptide, the fragmentation pattern, and the detected fragment ions are shown at the top right in each panel. b ions contain the N-terminal region of the peptide; y ions contain the C-terminal region of the peptide. Deamidation of asparagine to aspartic acid is designated as “ N ” with an underscore .

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    5) Product Images from "Integrin-Mediated Host Cell Invasion by Type 1-Piliated Uropathogenic Escherichia coli"

    Article Title: Integrin-Mediated Host Cell Invasion by Type 1-Piliated Uropathogenic Escherichia coli

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030100

    Glycosidase Treatment Abrogates In Vitro Binding of FimH to β1 and α3 Integrins (A) β1 and (B) α3 integrins were immunoprecipitated from 5637 cells that had been transiently transfected with plasmids for overexpression of recombinant human β1 or α3 integrins. The immunoprecipitated proteins were treated ± glycosidases (endoglycosidase Hf [EndoHf] or Peptide: N-glycosidase F [PNGase]) prior to SDS-PAGE and transfer to PVDF membranes. Blots were probed with either (A) anti–β1 or (B) anti–α3 integrin antibodies (Westerns), revealing expected shifts in the electrophoretic mobility of both integrin subunits following glycosidase treatments. For each set of samples, duplicate blots were overlaid with purified recombinant FimC 6XHisFLAG –FimH complexes and probed with anti-FLAG tag antibody (Far Westerns). As additional controls, blots containing untreated β1 or α3 integrins were also incubated with either FimC 6XHisFLAG –FimH complexes plus 2.5% D-mannose or with FimC 6XHisFLAG  alone. Molecular weight standards are indicated on the left.
    Figure Legend Snippet: Glycosidase Treatment Abrogates In Vitro Binding of FimH to β1 and α3 Integrins (A) β1 and (B) α3 integrins were immunoprecipitated from 5637 cells that had been transiently transfected with plasmids for overexpression of recombinant human β1 or α3 integrins. The immunoprecipitated proteins were treated ± glycosidases (endoglycosidase Hf [EndoHf] or Peptide: N-glycosidase F [PNGase]) prior to SDS-PAGE and transfer to PVDF membranes. Blots were probed with either (A) anti–β1 or (B) anti–α3 integrin antibodies (Westerns), revealing expected shifts in the electrophoretic mobility of both integrin subunits following glycosidase treatments. For each set of samples, duplicate blots were overlaid with purified recombinant FimC 6XHisFLAG –FimH complexes and probed with anti-FLAG tag antibody (Far Westerns). As additional controls, blots containing untreated β1 or α3 integrins were also incubated with either FimC 6XHisFLAG –FimH complexes plus 2.5% D-mannose or with FimC 6XHisFLAG alone. Molecular weight standards are indicated on the left.

    Techniques Used: In Vitro, Binding Assay, Immunoprecipitation, Transfection, Over Expression, Recombinant, SDS Page, Purification, FLAG-tag, Incubation, Molecular Weight

    6) Product Images from "Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion"

    Article Title: Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-04-0874

    Inversion and PM localization of marginally hydrophobic NA TMDs is C-tail-length dependent. (A) PM/IC ratios of cells expressing the marginally hydrophobic NA construct ( TM∆G +1.3 NA) with increasing C-tail lengths and representative cell section images. (B) Orientation of the marginally hydrophobic NA TMD construct with increasing C-tail lengths was determined by the expected N-linked glycosylation pattern (depicted by the construct diagrams) at steady state (top, immunoblots) and after metabolic labeling (bottom, [ 35 S]Met/Cys autoradiographs). Cell lysates were harvested 48 h posttransfection or after a 15-min pulse, and, where indicated, N-linked glycans were digested with PNGase F. The N-linked glycan number (arrowheads), partial PNGase F digestion (asterisks), and SDS-resistant TMD tetramer (Tet) are indicated. (C) The percent glycosylation of the marginally hydrophobic NA TMDs with respect to C-tail length from steady-state and 35 S-labeling experiments is shown. Glycosylation indicates that the constructs have the proper N in -C out orientation. (D) Analysis of the indicated 35 S-labeled constructs by nonreducing and reducing SDS–PAGE reveals that only the glycosylated species form the expected intermolecular disulfide. Note the glycosylated-band molecular weight shifts ± DTT and the partial SDS-resistant tetrameric species.
    Figure Legend Snippet: Inversion and PM localization of marginally hydrophobic NA TMDs is C-tail-length dependent. (A) PM/IC ratios of cells expressing the marginally hydrophobic NA construct ( TM∆G +1.3 NA) with increasing C-tail lengths and representative cell section images. (B) Orientation of the marginally hydrophobic NA TMD construct with increasing C-tail lengths was determined by the expected N-linked glycosylation pattern (depicted by the construct diagrams) at steady state (top, immunoblots) and after metabolic labeling (bottom, [ 35 S]Met/Cys autoradiographs). Cell lysates were harvested 48 h posttransfection or after a 15-min pulse, and, where indicated, N-linked glycans were digested with PNGase F. The N-linked glycan number (arrowheads), partial PNGase F digestion (asterisks), and SDS-resistant TMD tetramer (Tet) are indicated. (C) The percent glycosylation of the marginally hydrophobic NA TMDs with respect to C-tail length from steady-state and 35 S-labeling experiments is shown. Glycosylation indicates that the constructs have the proper N in -C out orientation. (D) Analysis of the indicated 35 S-labeled constructs by nonreducing and reducing SDS–PAGE reveals that only the glycosylated species form the expected intermolecular disulfide. Note the glycosylated-band molecular weight shifts ± DTT and the partial SDS-resistant tetrameric species.

    Techniques Used: Expressing, Construct, Western Blot, Labeling, SDS Page, Molecular Weight

    NA TMDs with a short C-tail have a stricter hydrophobicity requirement. (A) PM/IC ratios of individual cells expressing NA with a short, 36-aa C-tail and TMDs that range from being marginally hydrophobic (+1.3 kcal/mol) to hydrophobic (−2.5 kcal/mol). The immunoblot shows the constructs that received the expected N-linked glycan (arrowhead), which was confirmed by digestion with PNGase F. (B) Representative cell section images of the constructs analyzed in A. Inset, the mislocalized  TM∆G +1.3 NA 36aa  (green) with respect to the nucleus (blue).
    Figure Legend Snippet: NA TMDs with a short C-tail have a stricter hydrophobicity requirement. (A) PM/IC ratios of individual cells expressing NA with a short, 36-aa C-tail and TMDs that range from being marginally hydrophobic (+1.3 kcal/mol) to hydrophobic (−2.5 kcal/mol). The immunoblot shows the constructs that received the expected N-linked glycan (arrowhead), which was confirmed by digestion with PNGase F. (B) Representative cell section images of the constructs analyzed in A. Inset, the mislocalized TM∆G +1.3 NA 36aa (green) with respect to the nucleus (blue).

    Techniques Used: Expressing, Construct

    Hydrophobic NA TMDs are less dependent on C-tail length for inversion. (A) The orientation of the hydrophobic NA TMD construct with increasing C-tail lengths was analyzed at steady-state (top, immunoblots) and after metabolic labeling for 15 min (bottom, [ 35 S]Met/Cys autoradiographs) as described in Figure 4B . The N-linked glycan number (arrowheads) and partial PNGase F digested species (asterisk) are shown. (B) The percent glycosylation observed for the hydrophobic NA TMDs with respect to C-tail length from steady-state and 35 S-labeling experiments is shown.
    Figure Legend Snippet: Hydrophobic NA TMDs are less dependent on C-tail length for inversion. (A) The orientation of the hydrophobic NA TMD construct with increasing C-tail lengths was analyzed at steady-state (top, immunoblots) and after metabolic labeling for 15 min (bottom, [ 35 S]Met/Cys autoradiographs) as described in Figure 4B . The N-linked glycan number (arrowheads) and partial PNGase F digested species (asterisk) are shown. (B) The percent glycosylation observed for the hydrophobic NA TMDs with respect to C-tail length from steady-state and 35 S-labeling experiments is shown.

    Techniques Used: Construct, Western Blot, Labeling

    An elongated C-tail can invert the N out -C in  M2 TMD and is associated with marginally hydrophobic human N in -C out  (type II) TMDs. (A) Immunoblot of reduced and nonreduced cell lysates showing the N out -C in  orientation of M2 based on the intermolecular disulfide bonds formed by its N-terminal Cys residues in the ER lumen. (B) Immunoblots of untreated and PNGase F–treated lysates showing the glycosylation patterns for  TMΔG+1.3 NA 76aa , M2 with the 76-aa NA C-tail, full-length NA ( TMΔG+1.3 NA 440aa ), and M2 with the full-length 440-aa NA C-tail with the enzymatic domain. The number of N-linked glycans for each species is indicated. (C) Enzymatic activity was used to confirm M2 TMD inversion, as NA only folds within the ER lumen. The activity rate of M2 with the full-length 440-aa NA C-tail was calculated in comparison to lysates from cells expressing full-length NA as described in   da Silva   et al.  (2013) . (D) TMD hydrophobicity for the annotated type II human membrane proteins with respect to the length of the C-terminus after their TMD (C-tail). Dashed line at 100 aa corresponds to the ∼50% inversion point for newly synthesized NA with a marginally hydrophobic TMD. Regions covering the marginally hydrophobic TMDs and potential tail-anchored pathway substrates are highlighted. Protein sequences were obtained from Uniprot and analyzed using MPEx. The raw data and references are given in Supplemental Table S1.
    Figure Legend Snippet: An elongated C-tail can invert the N out -C in M2 TMD and is associated with marginally hydrophobic human N in -C out (type II) TMDs. (A) Immunoblot of reduced and nonreduced cell lysates showing the N out -C in orientation of M2 based on the intermolecular disulfide bonds formed by its N-terminal Cys residues in the ER lumen. (B) Immunoblots of untreated and PNGase F–treated lysates showing the glycosylation patterns for TMΔG+1.3 NA 76aa , M2 with the 76-aa NA C-tail, full-length NA ( TMΔG+1.3 NA 440aa ), and M2 with the full-length 440-aa NA C-tail with the enzymatic domain. The number of N-linked glycans for each species is indicated. (C) Enzymatic activity was used to confirm M2 TMD inversion, as NA only folds within the ER lumen. The activity rate of M2 with the full-length 440-aa NA C-tail was calculated in comparison to lysates from cells expressing full-length NA as described in da Silva et al. (2013) . (D) TMD hydrophobicity for the annotated type II human membrane proteins with respect to the length of the C-terminus after their TMD (C-tail). Dashed line at 100 aa corresponds to the ∼50% inversion point for newly synthesized NA with a marginally hydrophobic TMD. Regions covering the marginally hydrophobic TMDs and potential tail-anchored pathway substrates are highlighted. Protein sequences were obtained from Uniprot and analyzed using MPEx. The raw data and references are given in Supplemental Table S1.

    Techniques Used: Western Blot, Activity Assay, Expressing, Synthesized

    N-terminal flanking residues help marginally hydrophobic NA TMDs to invert. (A) Logo plot displaying the conservation of the cytoplasmic-localized, N-terminal NA TMD flanking residues in the human H1N1 IAV sequences. (B) Diagram of the N-terminal mutations and deletions that were analyzed in the TMΔG+1.3 NA 76aa construct. (C) Immunoblots showing the orientation of the constructs depicted in B by the absence or presence of the expected two N-linked glycans (arrowheads). Cell lysates were harvested 48 h posttransfection, and a portion was treated with PNGase F before resolution by reducing Tris-tricine SDS–PAGE. (D) PM/IC ratios from cells expressing the constructs shown in B with representative cell section images. Insets, cellular localization of the constructs (green) with respect to the nucleus (blue).
    Figure Legend Snippet: N-terminal flanking residues help marginally hydrophobic NA TMDs to invert. (A) Logo plot displaying the conservation of the cytoplasmic-localized, N-terminal NA TMD flanking residues in the human H1N1 IAV sequences. (B) Diagram of the N-terminal mutations and deletions that were analyzed in the TMΔG+1.3 NA 76aa construct. (C) Immunoblots showing the orientation of the constructs depicted in B by the absence or presence of the expected two N-linked glycans (arrowheads). Cell lysates were harvested 48 h posttransfection, and a portion was treated with PNGase F before resolution by reducing Tris-tricine SDS–PAGE. (D) PM/IC ratios from cells expressing the constructs shown in B with representative cell section images. Insets, cellular localization of the constructs (green) with respect to the nucleus (blue).

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

    7) Product Images from "Toward developing recombinant gonadotropin-based hormone therapies for increasing fertility in the flatfish Senegalese sole"

    Article Title: Toward developing recombinant gonadotropin-based hormone therapies for increasing fertility in the flatfish Senegalese sole

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174387

    Characterization of Senegalese sole recombinant gonadotropins and half-life of the hormones in plasma. (A, B) Characterization of CHO cells-expressed recombinant single-chain Fsh and Lh (rFsh and rLh) by Western blot analysis and deglycosylation. Concentrated supernatants of cell cultures were separated by 12% SDS-PAGE and immunoreacted with antibodies against Senegalese sole Fsh and Lh β subunits. For deglycosylation, denatured and reduced proteins were incubated with (+) or without (-) N-glycosidase F (PNGase F). Molecular mass markers (kDa) are on the left. (C, D) Time-course of plasma Fsh and Lh concentrations in fish (mean ± SEM;  n  = 5 fish) after intramuscular injection of saline or 6 μg/fish (17.5 ± 0.6 μg/kg) of rFsh or rLh. *,  P
    Figure Legend Snippet: Characterization of Senegalese sole recombinant gonadotropins and half-life of the hormones in plasma. (A, B) Characterization of CHO cells-expressed recombinant single-chain Fsh and Lh (rFsh and rLh) by Western blot analysis and deglycosylation. Concentrated supernatants of cell cultures were separated by 12% SDS-PAGE and immunoreacted with antibodies against Senegalese sole Fsh and Lh β subunits. For deglycosylation, denatured and reduced proteins were incubated with (+) or without (-) N-glycosidase F (PNGase F). Molecular mass markers (kDa) are on the left. (C, D) Time-course of plasma Fsh and Lh concentrations in fish (mean ± SEM; n = 5 fish) after intramuscular injection of saline or 6 μg/fish (17.5 ± 0.6 μg/kg) of rFsh or rLh. *, P

    Techniques Used: Recombinant, Western Blot, SDS Page, Incubation, Fluorescence In Situ Hybridization, Injection

    8) Product Images from "Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2"

    Article Title: Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.6b00738

    Murine VEGFR-2 (FLK-1) is Highly Glycosylated A. Lysate from porcine aortic endothelial (PAE) cells with ectopic expression of murine VEGFR-2 (FLK-1) without treatment (−), treated with heat denatured PNGase F (Denat.), or treated with 500 units of PNGase F (+). B. Schematic of experimental workflow for the analysis of VEGFR-2 N -glycosylation. C. Schematic of murine VEGFR-2, with the seven immunogloubulin (Ig)-like domains in the ectodomain, the transmembrane (TM) domain, and the cytoplasmic domain (not to scale). N -glycosylation sequons are marked with circles and labeled according to their location in the sequence.
    Figure Legend Snippet: Murine VEGFR-2 (FLK-1) is Highly Glycosylated A. Lysate from porcine aortic endothelial (PAE) cells with ectopic expression of murine VEGFR-2 (FLK-1) without treatment (−), treated with heat denatured PNGase F (Denat.), or treated with 500 units of PNGase F (+). B. Schematic of experimental workflow for the analysis of VEGFR-2 N -glycosylation. C. Schematic of murine VEGFR-2, with the seven immunogloubulin (Ig)-like domains in the ectodomain, the transmembrane (TM) domain, and the cytoplasmic domain (not to scale). N -glycosylation sequons are marked with circles and labeled according to their location in the sequence.

    Techniques Used: Expressing, Labeling, Sequencing

    9) Product Images from "Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids"

    Article Title: Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids

    Journal: Innate immunity

    doi: 10.1177/1753425914524077

    hTLR2ED expression and purification from insect cell culture A) Culture supernatants of insect cells infected with hTLR2ED Profold ER1 were concentrated and purified to homogeneity by Ni-NTA affinity, anion exchange and gel filtration chromatography. Samples were analyzed on 4–20% SDS-PAGE and stained with Coomassie Blue. B) hTLR2ED is a monomer in solution. Purified hTLR2ED and molecular weight standards were run on a Superdex 200 10/30 gel filtration column. Purified hTLR2 ED elutes at 14.2 ml, which corresponds to the elution volume of a protein of 70,000 Da, which is approximately the molecular mass of the hTLR2ED monomer(n.b. the molecular weight of the hTLR2ED monomer as determined by MALDI TOF mass spectrometry is 69,903 Da). C) hTLR2ED has N-linked glycosylation. hTLR2 undigested (1) and digested overnight with PNGase F in native conditions (2). D–E) hTLR2ED contains intramolecular disulfide bonds. D) Purified hTLR2ED was run under reducing (1R) and non-reducing (2NR) conditions in an SDS PAGE gel. The difference in electrophoretic mobility indicates the presence of disulfide bridges . E) hTLR2ED was digested overnight at 37°C with PNGase F and Endo H and then run in SDS PAGE under reducing (1R) and non-reducing (2NR) conditions (E). Molecular weight markers are shown on each gel in kDa.
    Figure Legend Snippet: hTLR2ED expression and purification from insect cell culture A) Culture supernatants of insect cells infected with hTLR2ED Profold ER1 were concentrated and purified to homogeneity by Ni-NTA affinity, anion exchange and gel filtration chromatography. Samples were analyzed on 4–20% SDS-PAGE and stained with Coomassie Blue. B) hTLR2ED is a monomer in solution. Purified hTLR2ED and molecular weight standards were run on a Superdex 200 10/30 gel filtration column. Purified hTLR2 ED elutes at 14.2 ml, which corresponds to the elution volume of a protein of 70,000 Da, which is approximately the molecular mass of the hTLR2ED monomer(n.b. the molecular weight of the hTLR2ED monomer as determined by MALDI TOF mass spectrometry is 69,903 Da). C) hTLR2ED has N-linked glycosylation. hTLR2 undigested (1) and digested overnight with PNGase F in native conditions (2). D–E) hTLR2ED contains intramolecular disulfide bonds. D) Purified hTLR2ED was run under reducing (1R) and non-reducing (2NR) conditions in an SDS PAGE gel. The difference in electrophoretic mobility indicates the presence of disulfide bridges . E) hTLR2ED was digested overnight at 37°C with PNGase F and Endo H and then run in SDS PAGE under reducing (1R) and non-reducing (2NR) conditions (E). Molecular weight markers are shown on each gel in kDa.

    Techniques Used: Expressing, Purification, Cell Culture, Infection, Filtration, Chromatography, SDS Page, Staining, Molecular Weight, Mass Spectrometry

    10) Product Images from "Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition *"

    Article Title: Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.085472

    gp120 glycosylation is influenced by metabolic manipulations. Jurkat and HepG2 cells stably transfected with gp120-encoding DNA plasmid were cultured in the presence of N -acetylglucosamine, 80 m m (GlcNAc); uridine, 5 m m ( Uri ); N -acetylglucosamine 80 m m plus uridine 5 m m (Uri+GlcNAc); succinate, 20 m m (S), pyruvate, 4.5 m m (Pyr); Gal, 50 m m ; Man, 50 m m ; DMJM, 800 μ m ; or mock-treated ( Untr , negative control). gp120 was purified using Ni-NTA-agarose, separated by 10% SDS-PAGE, blotted, and developed with anti-V5-tag antibody ( A ). Purified gp120 glycoproteins were treated with PNGase F (*), Endo H (**), or mock treated (***), and the resultant preparations were analyzed by SDS-PAGE and Western blotting with anti-V5-tag antibody. gp120 was from Jurkat ( B ) and HepG2 cells ( C ). Representative results from two experiments are shown.
    Figure Legend Snippet: gp120 glycosylation is influenced by metabolic manipulations. Jurkat and HepG2 cells stably transfected with gp120-encoding DNA plasmid were cultured in the presence of N -acetylglucosamine, 80 m m (GlcNAc); uridine, 5 m m ( Uri ); N -acetylglucosamine 80 m m plus uridine 5 m m (Uri+GlcNAc); succinate, 20 m m (S), pyruvate, 4.5 m m (Pyr); Gal, 50 m m ; Man, 50 m m ; DMJM, 800 μ m ; or mock-treated ( Untr , negative control). gp120 was purified using Ni-NTA-agarose, separated by 10% SDS-PAGE, blotted, and developed with anti-V5-tag antibody ( A ). Purified gp120 glycoproteins were treated with PNGase F (*), Endo H (**), or mock treated (***), and the resultant preparations were analyzed by SDS-PAGE and Western blotting with anti-V5-tag antibody. gp120 was from Jurkat ( B ) and HepG2 cells ( C ). Representative results from two experiments are shown.

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Negative Control, Purification, SDS Page, Western Blot

    Examples of N -glycan profiles from MALDI-TOF mass spectra. N -Linked oligosaccharides were isolated after digestion of gp120 expressed by HepG2 ( A ) or Jurkat cell lines ( B ) with PNGase F. Symbols with numbers indicate the number of saccharides added to the common core (GlcNAc) 2 (Man) 3 . Samples were prepared with PNGase F enzyme isolated from Flavobacterium that contained trace amounts of endo- and exoglycosidases that resulted in removing the reducing-end GlcNAc from high-mannose glycans (see “Experimental Procedures” for details). Identities of these glycans were confirmed by tandem mass spectrometry with LTQ mass spectrometry. *, denotes high-mannose glycans with (GlcNAc) 1 (Man) 3 core. Representative results from two experiments are shown. Supplemental Fig. S3 shows MALDI-TOF mass spectra of glycans of the five gp120 preparations with molecular masses and compositions of detected glycans indicated.
    Figure Legend Snippet: Examples of N -glycan profiles from MALDI-TOF mass spectra. N -Linked oligosaccharides were isolated after digestion of gp120 expressed by HepG2 ( A ) or Jurkat cell lines ( B ) with PNGase F. Symbols with numbers indicate the number of saccharides added to the common core (GlcNAc) 2 (Man) 3 . Samples were prepared with PNGase F enzyme isolated from Flavobacterium that contained trace amounts of endo- and exoglycosidases that resulted in removing the reducing-end GlcNAc from high-mannose glycans (see “Experimental Procedures” for details). Identities of these glycans were confirmed by tandem mass spectrometry with LTQ mass spectrometry. *, denotes high-mannose glycans with (GlcNAc) 1 (Man) 3 core. Representative results from two experiments are shown. Supplemental Fig. S3 shows MALDI-TOF mass spectra of glycans of the five gp120 preparations with molecular masses and compositions of detected glycans indicated.

    Techniques Used: Isolation, Mass Spectrometry

    Reactivity of selected lectins with N -linked oligosaccharides. Scheme of reactivity of gp120 after digestion with PNGase F (to remove all N -linked oligosaccharides), Endo H (to cleave only high-mannose and hybrid oligosaccharides), or neuraminidase (to remove sialic acid). Reactivities of lectins GNL, AAL, PHA-L, and Sambucus nigra agglutinin ( SNA ) with high-mannose or hybrid ( A ) and complex ( B ) oligosaccharides are based on recognition of specific oligosaccharide structures remaining on gp120 after treatment with specified glycosidase. For simplicity, the reactivity is expressed as plus (+) or minus (−).
    Figure Legend Snippet: Reactivity of selected lectins with N -linked oligosaccharides. Scheme of reactivity of gp120 after digestion with PNGase F (to remove all N -linked oligosaccharides), Endo H (to cleave only high-mannose and hybrid oligosaccharides), or neuraminidase (to remove sialic acid). Reactivities of lectins GNL, AAL, PHA-L, and Sambucus nigra agglutinin ( SNA ) with high-mannose or hybrid ( A ) and complex ( B ) oligosaccharides are based on recognition of specific oligosaccharide structures remaining on gp120 after treatment with specified glycosidase. For simplicity, the reactivity is expressed as plus (+) or minus (−).

    Techniques Used:

    Analysis of gp120 glycans by mobility shift after endoglycosidase treatment.  gp120 expressed in 293T, Jurkat, Dakiki, RD, HepG2, and CHO cell lines were treated with PNGase F, Endo H, or neuraminidase or remained untreated after which the preparations were separated by SDS-PAGE under reducing conditions, blotted on polyvinylidene difluoride membrane, and developed with anti-V5-tag antibody ( A ), lectins  G. nivalis ,  GNL  (high-mannose glycan-specific) ( B ),  A. aurantia ,  AAL  (fucose-specific) ( C ), or  P. vulgaris ,  PHA-L  (specific for complex glycans with ≥3 antennas) ( D ). Representative data from at least two different experiments are shown.
    Figure Legend Snippet: Analysis of gp120 glycans by mobility shift after endoglycosidase treatment. gp120 expressed in 293T, Jurkat, Dakiki, RD, HepG2, and CHO cell lines were treated with PNGase F, Endo H, or neuraminidase or remained untreated after which the preparations were separated by SDS-PAGE under reducing conditions, blotted on polyvinylidene difluoride membrane, and developed with anti-V5-tag antibody ( A ), lectins G. nivalis , GNL (high-mannose glycan-specific) ( B ), A. aurantia , AAL (fucose-specific) ( C ), or P. vulgaris , PHA-L (specific for complex glycans with ≥3 antennas) ( D ). Representative data from at least two different experiments are shown.

    Techniques Used: Mobility Shift, SDS Page

    11) Product Images from "Development of an Influenza A Master Virus for Generating High-Growth Reassortants for A/Anhui/1/2013(H7N9) Vaccine Production in Qualified MDCK Cells"

    Article Title: Development of an Influenza A Master Virus for Generating High-Growth Reassortants for A/Anhui/1/2013(H7N9) Vaccine Production in Qualified MDCK Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0160040

    Western blotting analysis of the purified viral proteins. Purified viral concentrates of NIIDRG-10C, -10.1C, -10 and -10.1 were analyzed by SDS-PAGE. HA proteins were detected using a rabbit polyclonal antibody against recombinant HA protein of H7N9 (A/Shanghai/1/2013) (Sino Biological Inc. Beijing, China) and a donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody by western blotting analysis. Purified viral proteins were treated (A) or untreated (B) with N-glycosidase F.
    Figure Legend Snippet: Western blotting analysis of the purified viral proteins. Purified viral concentrates of NIIDRG-10C, -10.1C, -10 and -10.1 were analyzed by SDS-PAGE. HA proteins were detected using a rabbit polyclonal antibody against recombinant HA protein of H7N9 (A/Shanghai/1/2013) (Sino Biological Inc. Beijing, China) and a donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody by western blotting analysis. Purified viral proteins were treated (A) or untreated (B) with N-glycosidase F.

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

    12) Product Images from "Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes"

    Article Title: Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.172007

    Tsetse salivary glycans are recognized by C-type lectins Mannose Receptor and DC-SIGN. 2 μg of Glossina morsitans saliva ( Gmm ) were untreated (-) or treated (+) with PNGase F and then processed for overlay assays using either recombinant CTLD4-7-Fc (A) or DC-SIGN (B). MWM, lanes 1 and 7; Gmm saliva, lanes 2, 3, 8 and 9; OVA, egg albumin positive control (lanes 4, 5, 10 and 11); BSA, bovine serum albumin negative control (lanes 6 and 12). Nigrosine-stained membranes (B, D) are shown as loading controls for (A) and (B), respectively. Asterisk indicates PNGase F enzyme.
    Figure Legend Snippet: Tsetse salivary glycans are recognized by C-type lectins Mannose Receptor and DC-SIGN. 2 μg of Glossina morsitans saliva ( Gmm ) were untreated (-) or treated (+) with PNGase F and then processed for overlay assays using either recombinant CTLD4-7-Fc (A) or DC-SIGN (B). MWM, lanes 1 and 7; Gmm saliva, lanes 2, 3, 8 and 9; OVA, egg albumin positive control (lanes 4, 5, 10 and 11); BSA, bovine serum albumin negative control (lanes 6 and 12). Nigrosine-stained membranes (B, D) are shown as loading controls for (A) and (B), respectively. Asterisk indicates PNGase F enzyme.

    Techniques Used: Recombinant, Positive Control, Negative Control, Staining

    Analysis of the effects of infection on immunogenicity of tsetse fly saliva. (A) 2 μg of G. m. morsitans salivary proteins were treated (+) or untreated (-) with PNGase F, fractionated by SDS-PAGE, transferred onto a PVDF membrane, and probed with an anti- G. m. morsitans saliva antibody. (B) Uniform protein loading for Western blot was confirmed by nigrosine staining of proteins transferred to PVDF membrane. (C) Con A blotting analysis of tsetse salivary glycoproteins from naïve and trypanosome-infected flies. OVA, egg albumin positive control. Asterisk indicates PNGase F enzyme band.
    Figure Legend Snippet: Analysis of the effects of infection on immunogenicity of tsetse fly saliva. (A) 2 μg of G. m. morsitans salivary proteins were treated (+) or untreated (-) with PNGase F, fractionated by SDS-PAGE, transferred onto a PVDF membrane, and probed with an anti- G. m. morsitans saliva antibody. (B) Uniform protein loading for Western blot was confirmed by nigrosine staining of proteins transferred to PVDF membrane. (C) Con A blotting analysis of tsetse salivary glycoproteins from naïve and trypanosome-infected flies. OVA, egg albumin positive control. Asterisk indicates PNGase F enzyme band.

    Techniques Used: Infection, SDS Page, Western Blot, Staining, Positive Control

    Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose glycans. [A] Profile of salivary  N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled  N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH,  Streptococcus pneumonia  in  E. coli  β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analysed by HILIC-(U)HPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different glycans are listed in   Table 1 . [B] Positive-ion ESI-MS spectrum of procainamide-labelled  N -glycans from teneral tsetse fly saliva. Numbers refer to the structures shown in   Table 1 . Asterisk (*) refers to  m/z  1130.55 as [M+2H] 2+  ion. Green circle, mannose; blue square,  N -Acetylglucosamine; red triangle, fucose; Proc, procainamide.
    Figure Legend Snippet: Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose glycans. [A] Profile of salivary N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumonia in E. coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analysed by HILIC-(U)HPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different glycans are listed in Table 1 . [B] Positive-ion ESI-MS spectrum of procainamide-labelled N -glycans from teneral tsetse fly saliva. Numbers refer to the structures shown in Table 1 . Asterisk (*) refers to m/z 1130.55 as [M+2H] 2+ ion. Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide.

    Techniques Used: Labeling, Incubation, Hydrophilic Interaction Liquid Chromatography

    Analysis of tsetse salivary  N -linked glycans in teneral, naïve and trypanosome-infected flies. [A] Comparison of HILIC-(U)HPLC profiles of salivary  N -glycans released by PNGase F. Analysis of 2AB-labelled glycans from (i) teneral, (ii) naïve, and (iii) trypanosome-infected saliva. Relative abundances are indicated in   table 2 . Tbb,  Trypanosoma brucei brucei.  [B] Positive-ion ESIMS analysis of procainamide labelled  M- glycans from adult naïve and trypanosome-infected saliva. Spectra are shown for naïve (top) and trypanosome-infected (bottom) saliva. Numbers refer to the structures shown in   Table 1 . Green circle, mannose; blue square,  N -Acetylglucosamine; red triangle, fucose; Proc, procainamide.
    Figure Legend Snippet: Analysis of tsetse salivary N -linked glycans in teneral, naïve and trypanosome-infected flies. [A] Comparison of HILIC-(U)HPLC profiles of salivary N -glycans released by PNGase F. Analysis of 2AB-labelled glycans from (i) teneral, (ii) naïve, and (iii) trypanosome-infected saliva. Relative abundances are indicated in table 2 . Tbb, Trypanosoma brucei brucei. [B] Positive-ion ESIMS analysis of procainamide labelled M- glycans from adult naïve and trypanosome-infected saliva. Spectra are shown for naïve (top) and trypanosome-infected (bottom) saliva. Numbers refer to the structures shown in Table 1 . Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide.

    Techniques Used: Infection, Hydrophilic Interaction Liquid Chromatography

    13) Product Images from "Characterization and Immunotherapeutic Implications for a Novel Antibody Targeting Interleukin (IL)-13 Receptor ?2 *"

    Article Title: Characterization and Immunotherapeutic Implications for a Novel Antibody Targeting Interleukin (IL)-13 Receptor ?2 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.370015

    Effect of N -linked glycosylation on the binding of IL13Rα2 to recombinant IL13Rα2. A , binding of IL13Rα2 to control and Pngase F-treated rhIL13Rα2. Plates were coated with hrIL13Rα2 at 1 μg/ml and treated with native buffer or with 1 milliunit/well Pngase F in native buffer for 3 h at 37 °C. An ELISA for binding of the IL13Rα2 (clone 47) mAb in comparison with antibody clones B-D13, 83807, and YY-23Z and rhIL-13 was performed, and the data of one representative experiment from three independent experiments are shown. A paired t test was used to evaluate the difference between control and Pngase F-treated groups ( n = 4). *, p
    Figure Legend Snippet: Effect of N -linked glycosylation on the binding of IL13Rα2 to recombinant IL13Rα2. A , binding of IL13Rα2 to control and Pngase F-treated rhIL13Rα2. Plates were coated with hrIL13Rα2 at 1 μg/ml and treated with native buffer or with 1 milliunit/well Pngase F in native buffer for 3 h at 37 °C. An ELISA for binding of the IL13Rα2 (clone 47) mAb in comparison with antibody clones B-D13, 83807, and YY-23Z and rhIL-13 was performed, and the data of one representative experiment from three independent experiments are shown. A paired t test was used to evaluate the difference between control and Pngase F-treated groups ( n = 4). *, p

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Clone Assay

    14) Product Images from "Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin"

    Article Title: Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin

    Journal: Vaccines

    doi: 10.3390/vaccines8040781

    SDS-PAGE of purified total viral protein of recombinant H5N6 viruses. Purified total viral protein of rH5N6-310PB2 (lane 1, 3) and rH5N6-H103Y-310PB2 (lane 2, 4) acquired by ultracentrifugation were separated by SDS-PAGE. Deglycosylation of viral proteins was conducted using PNGase F and deglycosylated protein were also separated by SDS-PAGE (lane 3, 4). Lane 1; total viral protein of rH5N6-310PB2, lane 2; total viral protein of rH5N6-H103Y-310PB2, lane 3; PNGase F treated total viral protein of rH5N6-310PB2 and lane 4; PNGase F treated total viral protein of rH5N6- H103Y-310PB2.
    Figure Legend Snippet: SDS-PAGE of purified total viral protein of recombinant H5N6 viruses. Purified total viral protein of rH5N6-310PB2 (lane 1, 3) and rH5N6-H103Y-310PB2 (lane 2, 4) acquired by ultracentrifugation were separated by SDS-PAGE. Deglycosylation of viral proteins was conducted using PNGase F and deglycosylated protein were also separated by SDS-PAGE (lane 3, 4). Lane 1; total viral protein of rH5N6-310PB2, lane 2; total viral protein of rH5N6-H103Y-310PB2, lane 3; PNGase F treated total viral protein of rH5N6-310PB2 and lane 4; PNGase F treated total viral protein of rH5N6- H103Y-310PB2.

    Techniques Used: SDS Page, Purification, Recombinant

    15) Product Images from "The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu"

    Article Title: The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

    Journal: Retrovirology

    doi: 10.1186/1742-4690-6-80

    Comparison of endogenous BST-2 in HeLa cells to BST-2 expressed in transiently transfected 293T cells . (A) 293T cells were transfected with wt BST-2 (lane 3). A mock transfected culture from HeLa (lane 1) and 293T cells (lane 2) was analyzed in parallel. Whole cell lysates were processed for immunoblotting as described in Methods. The arrow identifies a BST-2 species in transfected 293T cells not seen in HeLa cells. (B C) Endoglycosidase analysis of transiently expressed BST-2. 293T cells were transfected with pcDNA-BST-2. BST-2 was enriched by adsorption to either datura stramonium lectin resin (DS lectin) (B) or Concanavalin A resin (ConA) (C) as described in Methods. DS lectin or ConA bound proteins were either left untreated (lanes 1 4) or treated with endoglycosidase H (EndoH) (lanes 2), Peptide: N-Glycosidase F (PNGase) (lanes 3), or endo-β-galactosidase (EndoB) (lanes 5) as described in Methods. Proteins were visualized by immunoblot analysis using a BST-2 specific antibody. (D) HeLa extracts were adsorbed to DS lectin (lane 2) and ConA resin (lane 3) as described for panels B C. Total input lysate is shown in lane 1. A high mannose form of endogenous BST-2 was enriched on the ConA resin.
    Figure Legend Snippet: Comparison of endogenous BST-2 in HeLa cells to BST-2 expressed in transiently transfected 293T cells . (A) 293T cells were transfected with wt BST-2 (lane 3). A mock transfected culture from HeLa (lane 1) and 293T cells (lane 2) was analyzed in parallel. Whole cell lysates were processed for immunoblotting as described in Methods. The arrow identifies a BST-2 species in transfected 293T cells not seen in HeLa cells. (B C) Endoglycosidase analysis of transiently expressed BST-2. 293T cells were transfected with pcDNA-BST-2. BST-2 was enriched by adsorption to either datura stramonium lectin resin (DS lectin) (B) or Concanavalin A resin (ConA) (C) as described in Methods. DS lectin or ConA bound proteins were either left untreated (lanes 1 4) or treated with endoglycosidase H (EndoH) (lanes 2), Peptide: N-Glycosidase F (PNGase) (lanes 3), or endo-β-galactosidase (EndoB) (lanes 5) as described in Methods. Proteins were visualized by immunoblot analysis using a BST-2 specific antibody. (D) HeLa extracts were adsorbed to DS lectin (lane 2) and ConA resin (lane 3) as described for panels B C. Total input lysate is shown in lane 1. A high mannose form of endogenous BST-2 was enriched on the ConA resin.

    Techniques Used: Transfection, Adsorption

    16) Product Images from "Three Amino Acid Changes in Avian Coronavirus Spike Protein Allow Binding to Kidney Tissue"

    Article Title: Three Amino Acid Changes in Avian Coronavirus Spike Protein Allow Binding to Kidney Tissue

    Journal: Journal of Virology

    doi: 10.1128/JVI.01363-19

    IBV M41- and QX-RBD protein analysis. (A) Amino acid alignment of M41-RBD (amino acids 19 to 272; GenBank accession number AY851295 ) and amino acids 19 to 275 (GenBank accession number AFJ11176 ) of the QX spike. Numbering starts at 1 of the mature protein sequence (signal sequence not shown). Dots indicate identical amino acids. Gray highlights surrounded by a black box indicate previously identified hypervariable regions of IBV-Mass ( 22 ). Green highlights indicate very different residues. (B) M41- and QX-RBD with and without pretreatment of PNGase F analyzed by Western blotting using Strep-Tactin HRP antibody. (C) Percentage of secondary protein structures calculated based on CD analysis of M41- and QX-RBD. (D) Binding of QX-RBD to paraffin-embedded healthy chicken trachea and kidney visualized by red staining in protein histochemistry.
    Figure Legend Snippet: IBV M41- and QX-RBD protein analysis. (A) Amino acid alignment of M41-RBD (amino acids 19 to 272; GenBank accession number AY851295 ) and amino acids 19 to 275 (GenBank accession number AFJ11176 ) of the QX spike. Numbering starts at 1 of the mature protein sequence (signal sequence not shown). Dots indicate identical amino acids. Gray highlights surrounded by a black box indicate previously identified hypervariable regions of IBV-Mass ( 22 ). Green highlights indicate very different residues. (B) M41- and QX-RBD with and without pretreatment of PNGase F analyzed by Western blotting using Strep-Tactin HRP antibody. (C) Percentage of secondary protein structures calculated based on CD analysis of M41- and QX-RBD. (D) Binding of QX-RBD to paraffin-embedded healthy chicken trachea and kidney visualized by red staining in protein histochemistry.

    Techniques Used: Sequencing, Western Blot, Binding Assay, Staining

    17) Product Images from "Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells"

    Article Title: Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells

    Journal: eLife

    doi: 10.7554/eLife.39494

    SPLTSS phosphorylation mediates MAVS degradation by RV infection. ( A ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( B ) Purified recombinant Flag-MAVS and RNaseB proteins were digested with PNGaseF or EndoH and measured by silver staining (upper panel) and western blot for potential AMPylation (lower panel). Top arrow marks recombinant MAVS protein (~72 kD) and bottom arrow marks recombinant RNaseB protein (~17 kD). ( C ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants (SPLTSS: SPLTSS mutated to six alanines; R3A: R232, R236, R239 mutated to three alanines) for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( D ) MAVS -/- HEK293 cells were transfected with WT or indicated MAVS mutants for 48 hr and harvested for RT-qPCR analysis measuring IFN-β and IFN-λ expression. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).
    Figure Legend Snippet: SPLTSS phosphorylation mediates MAVS degradation by RV infection. ( A ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( B ) Purified recombinant Flag-MAVS and RNaseB proteins were digested with PNGaseF or EndoH and measured by silver staining (upper panel) and western blot for potential AMPylation (lower panel). Top arrow marks recombinant MAVS protein (~72 kD) and bottom arrow marks recombinant RNaseB protein (~17 kD). ( C ) HEK293 cells were transfected with indicated Flag-tagged MAVS mutants (SPLTSS: SPLTSS mutated to six alanines; R3A: R232, R236, R239 mutated to three alanines) for 48 hr with or without human RV Wa infection (MOI = 3) for the last 12 hr. The levels of MAVS and GAPDH were measured by western blot. ( D ) MAVS -/- HEK293 cells were transfected with WT or indicated MAVS mutants for 48 hr and harvested for RT-qPCR analysis measuring IFN-β and IFN-λ expression. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).

    Techniques Used: Infection, Transfection, Western Blot, Purification, Recombinant, Silver Staining, Quantitative RT-PCR, Expressing

    18) Product Images from "N-Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of NaV1.5/β2 to the plasma membrane"

    Article Title: N-Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of NaV1.5/β2 to the plasma membrane

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.007903

    β2 is N -glycosylated at positions Asn-42, Asn-66, and Asn-74. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or partially or fully unglycosylated β2 or left untransfected ( utf ). A and B , cells were grown for 2 days in wells. Representative Western blots are shown with the same amount of protein lysate loaded into each lane. A , all glycosylation-defective mutants display increased electrophoretic mobility, with N42Q as the single mutant with the greatest change, and triple (fully) unglycosylated β2 showing complete shift. B , denatured protein from cell lysates was treated overnight at 37 °C with PNGase F to cleave off all N -glycans. C , cells were treated with TUN or with GalNAc- O -bn, to block N - or O -glycosylation, respectively, and grown for 1 day in wells; β2 WT remains unglycosylated only with TUN. DMSO , cells with the equivalent volume of solvent added; −, untreated cells. Blots for Na/K-ATPase or actin are included as loading controls. Molecular mass markers are in kDa.
    Figure Legend Snippet: β2 is N -glycosylated at positions Asn-42, Asn-66, and Asn-74. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or partially or fully unglycosylated β2 or left untransfected ( utf ). A and B , cells were grown for 2 days in wells. Representative Western blots are shown with the same amount of protein lysate loaded into each lane. A , all glycosylation-defective mutants display increased electrophoretic mobility, with N42Q as the single mutant with the greatest change, and triple (fully) unglycosylated β2 showing complete shift. B , denatured protein from cell lysates was treated overnight at 37 °C with PNGase F to cleave off all N -glycans. C , cells were treated with TUN or with GalNAc- O -bn, to block N - or O -glycosylation, respectively, and grown for 1 day in wells; β2 WT remains unglycosylated only with TUN. DMSO , cells with the equivalent volume of solvent added; −, untreated cells. Blots for Na/K-ATPase or actin are included as loading controls. Molecular mass markers are in kDa.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Blocking Assay

    19) Product Images from "Functional implications of corticosteroid-binding globulinN-glycosylation"

    Article Title: Functional implications of corticosteroid-binding globulinN-glycosylation

    Journal: Journal of Molecular Endocrinology

    doi: 10.1530/JME-17-0234

    Effects of inhibiting glycosylation or removing N -glycans on human and rat CBG steroid binding. The CBGs were expressed in CHO-S cells in the presence or absence of the N -glycosylation inhibitor tunicamycin (Tun) and the culture media were concentrated and buffer-exchanged for Western blotting and steroid-binding capacity measurements. Reductions in apparent molecular size and loss of micro-heterogeneity are consistent with the absence of glycosylation. The steroid-binding capacities of human and rat CBGs produced by tunicamycin-treated CHO-S cells were compared as a percentage (%) of those produced by untreated (Ctl) CHO-S cells after adjusting their amounts based on Western blotting. Similar amounts of human CBG, rat CBG, and human CBG N238 produced in untreated CHO-S cells were also incubated with PNGase F (PNG) to remove N -glycans. The amounts of PNGase F and incubation time were optimized to ensure that removal of N -glycans was as complete as possible, and similar results were observed when 500 units of PNGase F treatment were used for 3 h or 16 h at 37°C. Western blotting was used to assess the efficacy of deglycosylation and steroid-binding capacities were expressed as a percentage (%) of those obtained for the untreated (Ctl) samples. Positions of molecular size markers (kDa) are indicated.
    Figure Legend Snippet: Effects of inhibiting glycosylation or removing N -glycans on human and rat CBG steroid binding. The CBGs were expressed in CHO-S cells in the presence or absence of the N -glycosylation inhibitor tunicamycin (Tun) and the culture media were concentrated and buffer-exchanged for Western blotting and steroid-binding capacity measurements. Reductions in apparent molecular size and loss of micro-heterogeneity are consistent with the absence of glycosylation. The steroid-binding capacities of human and rat CBGs produced by tunicamycin-treated CHO-S cells were compared as a percentage (%) of those produced by untreated (Ctl) CHO-S cells after adjusting their amounts based on Western blotting. Similar amounts of human CBG, rat CBG, and human CBG N238 produced in untreated CHO-S cells were also incubated with PNGase F (PNG) to remove N -glycans. The amounts of PNGase F and incubation time were optimized to ensure that removal of N -glycans was as complete as possible, and similar results were observed when 500 units of PNGase F treatment were used for 3 h or 16 h at 37°C. Western blotting was used to assess the efficacy of deglycosylation and steroid-binding capacities were expressed as a percentage (%) of those obtained for the untreated (Ctl) samples. Positions of molecular size markers (kDa) are indicated.

    Techniques Used: Binding Assay, Western Blot, Produced, CTL Assay, Incubation

    20) Product Images from "Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells"

    Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgu164

    Purity and signaling transduction of rGEP. ( A ) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. ( B ) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP at 95% level according to Student’s t -test.
    Figure Legend Snippet: Purity and signaling transduction of rGEP. ( A ) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. ( B ) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP at 95% level according to Student’s t -test.

    Techniques Used: Transduction, Purification, SDS Page, Staining, Western Blot, Incubation, Positive Control, Activation Assay

    21) Product Images from "Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms"

    Article Title: Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.018960

    Interaction studies of the Ltbp-4L and Ltbp-4S N-terminal regions with full-length fibulin-4 and fibulin-5. (A) Domain structure of full-length Ltbp-4L and Ltbp-4S and the recombinantly expressed Ltbp-4L (Ltbp-4L-2xStrep) and Ltbp-4S (Ltbp-4S-2xStrep) N-terminal fragments. The full-length proteins consist of 4-cystein (4-cys) repeats (white rhombi), non-Ca 2+ -binding EGF-like repeats (black rectangles), Ca 2+ -binding EGF-like repeats (white rectangles), hybrid domains (black ellipses) and 8-cystein (8-cys) repeats (white ellipses). The N-terminal fragments consist of two (Ltbp-4L-2xStrep) or one (Ltbp-4S-2xStrep) unique 4-cys repeats, the common non-Ca 2+ -binding EGF-like repeat and a C-terminal 2xStrep tag (red ellipses). Binding sites for ECM proteins, putative N-glycosylation sites (green lines) as well as the amino acid (aa) lengths are indicated. (B,C) Sensorgrams from surface-plasmon resonance interaction experiments showed a stronger binding affinity of Ltbp-4L-2xStrep (0–320 nM) ‘flown’ over immobilized recombinant full-length fibulin-5 (B; rfibulin-5) or immobilized recombinant full-length fibulin-4 (C; rfibulin-4) compared to Ltbp-4S-2xStrep (0–80 nM) flown over immobilized rfibulin-5 (B) or rfibulin-4 (C). The results are expressed as resonance units (RUs; n =2). (D) Deglycosylation digest with PNGase F of denatured recombinant full-length human LTBP-4S (rLTBP-4S) showing that there is a shift towards lower molecular mass positions. (E) Upper panel: deglycosylation of Ltbp-4L-2xStrep and Ltbp-4S-2xStrep. Ltbp-4L-2xStrep was unaffected, whereas Ltbp-4S-2xStrep showed a shift towards lower molecular weight positions. Lower panel: Ltbp-4S-2xStrep was digested with PNGase F under native (left lanes) and denaturing (right lanes) conditions. Both conditions resulted in a shift towards lower molecular weight positions. (F,G) After digest under non-denaturing conditions Ltbp-4L-2xStrep and Ltbp-4S-2xStrep were both able to bind to rfibulin-4 and -5 immobilized on a Biacore chip. Ltbp-4L-2xStrep binding was not affected, whereas Ltbp-4S-2xStrep showed an increase in binding of 15% to 20% after deglycosylation. The continuous lines represented the response before and the dashed lines after deglycosylation ( n =2).
    Figure Legend Snippet: Interaction studies of the Ltbp-4L and Ltbp-4S N-terminal regions with full-length fibulin-4 and fibulin-5. (A) Domain structure of full-length Ltbp-4L and Ltbp-4S and the recombinantly expressed Ltbp-4L (Ltbp-4L-2xStrep) and Ltbp-4S (Ltbp-4S-2xStrep) N-terminal fragments. The full-length proteins consist of 4-cystein (4-cys) repeats (white rhombi), non-Ca 2+ -binding EGF-like repeats (black rectangles), Ca 2+ -binding EGF-like repeats (white rectangles), hybrid domains (black ellipses) and 8-cystein (8-cys) repeats (white ellipses). The N-terminal fragments consist of two (Ltbp-4L-2xStrep) or one (Ltbp-4S-2xStrep) unique 4-cys repeats, the common non-Ca 2+ -binding EGF-like repeat and a C-terminal 2xStrep tag (red ellipses). Binding sites for ECM proteins, putative N-glycosylation sites (green lines) as well as the amino acid (aa) lengths are indicated. (B,C) Sensorgrams from surface-plasmon resonance interaction experiments showed a stronger binding affinity of Ltbp-4L-2xStrep (0–320 nM) ‘flown’ over immobilized recombinant full-length fibulin-5 (B; rfibulin-5) or immobilized recombinant full-length fibulin-4 (C; rfibulin-4) compared to Ltbp-4S-2xStrep (0–80 nM) flown over immobilized rfibulin-5 (B) or rfibulin-4 (C). The results are expressed as resonance units (RUs; n =2). (D) Deglycosylation digest with PNGase F of denatured recombinant full-length human LTBP-4S (rLTBP-4S) showing that there is a shift towards lower molecular mass positions. (E) Upper panel: deglycosylation of Ltbp-4L-2xStrep and Ltbp-4S-2xStrep. Ltbp-4L-2xStrep was unaffected, whereas Ltbp-4S-2xStrep showed a shift towards lower molecular weight positions. Lower panel: Ltbp-4S-2xStrep was digested with PNGase F under native (left lanes) and denaturing (right lanes) conditions. Both conditions resulted in a shift towards lower molecular weight positions. (F,G) After digest under non-denaturing conditions Ltbp-4L-2xStrep and Ltbp-4S-2xStrep were both able to bind to rfibulin-4 and -5 immobilized on a Biacore chip. Ltbp-4L-2xStrep binding was not affected, whereas Ltbp-4S-2xStrep showed an increase in binding of 15% to 20% after deglycosylation. The continuous lines represented the response before and the dashed lines after deglycosylation ( n =2).

    Techniques Used: Binding Assay, SPR Assay, Recombinant, Molecular Weight, Chromatin Immunoprecipitation

    22) Product Images from "Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing"

    Article Title: Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129294

    SDS-PAGE and western blot analysis of gene expression in P . pastoris and transgenic maize. (A) SDS-PAGE analysis of recombinant Aga-F75 produced in P . pastoris . Lanes: 1, the purified Aga-F75 with PNGase F treatment; 2, the purified Aga-F75; M, the protein molecular markers. (B) Western blot analysis of Aga-F75M produced in transgenic maize. Lanes: 1, the protein extract of transgenic maize seeds with PNGase F treatment; 2, the protein extract of non-transgenic Zheng58 seeds as a negative control; M, the protein molecular markers; 3, the protein extract of transgenic maize seeds. (C) Specific expression of Aga-F75M in transgenic maize. Lanes: M, the protein molecular markers; 1, the purified recombinant Aga-F75 produced in P . pastoris ; 2, the protein extract of non-transgenic Zheng58 seeds; 3–5, the protein extracts of transgenic maize seeds; 6–8, the protein extracts of leaf, stem, and root of the transgenic plant, respectively.
    Figure Legend Snippet: SDS-PAGE and western blot analysis of gene expression in P . pastoris and transgenic maize. (A) SDS-PAGE analysis of recombinant Aga-F75 produced in P . pastoris . Lanes: 1, the purified Aga-F75 with PNGase F treatment; 2, the purified Aga-F75; M, the protein molecular markers. (B) Western blot analysis of Aga-F75M produced in transgenic maize. Lanes: 1, the protein extract of transgenic maize seeds with PNGase F treatment; 2, the protein extract of non-transgenic Zheng58 seeds as a negative control; M, the protein molecular markers; 3, the protein extract of transgenic maize seeds. (C) Specific expression of Aga-F75M in transgenic maize. Lanes: M, the protein molecular markers; 1, the purified recombinant Aga-F75 produced in P . pastoris ; 2, the protein extract of non-transgenic Zheng58 seeds; 3–5, the protein extracts of transgenic maize seeds; 6–8, the protein extracts of leaf, stem, and root of the transgenic plant, respectively.

    Techniques Used: SDS Page, Western Blot, Expressing, Transgenic Assay, Recombinant, Produced, Purification, Negative Control

    23) Product Images from "Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes"

    Article Title: Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0009071

    Tsetse salivary N -glycans are recognized by C-type lectins Mannose Receptor and DC-SIGN. 2 μg of Glossina morsitans saliva ( Gmm ) were untreated (-) or treated (+) with PNGase F and then processed for overlay assays using either recombinant CTLD4-7-Fc (A) or DC-SIGN (B). MWM, lanes 1 and 7; Gmm saliva, lanes 2, 3, 8 and 9; OVA, egg albumin positive control (lanes 4, 5, 10 and 11); BSA, bovine serum albumin negative control (lanes 6 and 12). Nigrosine-stained membranes (B, D) are shown as loading controls for (A) and (B), respectively. Asterisk indicates PNGase F enzyme.
    Figure Legend Snippet: Tsetse salivary N -glycans are recognized by C-type lectins Mannose Receptor and DC-SIGN. 2 μg of Glossina morsitans saliva ( Gmm ) were untreated (-) or treated (+) with PNGase F and then processed for overlay assays using either recombinant CTLD4-7-Fc (A) or DC-SIGN (B). MWM, lanes 1 and 7; Gmm saliva, lanes 2, 3, 8 and 9; OVA, egg albumin positive control (lanes 4, 5, 10 and 11); BSA, bovine serum albumin negative control (lanes 6 and 12). Nigrosine-stained membranes (B, D) are shown as loading controls for (A) and (B), respectively. Asterisk indicates PNGase F enzyme.

    Techniques Used: Recombinant, Positive Control, Negative Control, Staining

    Analysis of the effects of infection on immunogenicity of tsetse fly saliva. (A) 2 μg of G . m . morsitans salivary proteins were treated (+) or untreated (-) with PNGase F, fractionated by SDS-PAGE, transferred onto a PVDF membrane, and probed with an anti- G . m . morsitans saliva antibody. (B) Uniform protein loading for Western blot was confirmed by nigrosine staining of proteins transferred to PVDF membrane. (C) Con A blotting analysis of tsetse salivary glycoproteins from naïve and trypanosome-infected flies. OVA, egg albumin positive control. Asterisk indicates PNGase F enzyme band.
    Figure Legend Snippet: Analysis of the effects of infection on immunogenicity of tsetse fly saliva. (A) 2 μg of G . m . morsitans salivary proteins were treated (+) or untreated (-) with PNGase F, fractionated by SDS-PAGE, transferred onto a PVDF membrane, and probed with an anti- G . m . morsitans saliva antibody. (B) Uniform protein loading for Western blot was confirmed by nigrosine staining of proteins transferred to PVDF membrane. (C) Con A blotting analysis of tsetse salivary glycoproteins from naïve and trypanosome-infected flies. OVA, egg albumin positive control. Asterisk indicates PNGase F enzyme band.

    Techniques Used: Infection, SDS Page, Western Blot, Staining, Positive Control

    Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose  N -glycans. (A) Profile of salivary  N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled  N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH,  Streptococcus pneumoniae  in  E .  coli  β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analyzed by HILIC-UHPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different  N -glycans are listed in   Table 1 . (B) Positive-ion ESI-MS spectrum of procainamide-labelled  N -glycans from teneral tsetse fly saliva. Numbers refer to the structures in   Table 1 . The dagger symbol (‡) refers to  m/z  1130.55 as [M+2H] 2+  ion; the appearance of the Man 3 GlcNAc 2 -Proc as singly and doubly charged ion in positive mode, reflects on its high relative abundancy (~54%) in this sample. Green circle, mannose; blue square,  N -Acetylglucosamine; red triangle, fucose; Proc, procainamide. GU, glucose homopolymer ladder. [  22 ].
    Figure Legend Snippet: Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose N -glycans. (A) Profile of salivary N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumoniae in E . coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analyzed by HILIC-UHPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different N -glycans are listed in Table 1 . (B) Positive-ion ESI-MS spectrum of procainamide-labelled N -glycans from teneral tsetse fly saliva. Numbers refer to the structures in Table 1 . The dagger symbol (‡) refers to m/z 1130.55 as [M+2H] 2+ ion; the appearance of the Man 3 GlcNAc 2 -Proc as singly and doubly charged ion in positive mode, reflects on its high relative abundancy (~54%) in this sample. Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide. GU, glucose homopolymer ladder. [ 22 ].

    Techniques Used: Labeling, Incubation, Hydrophilic Interaction Liquid Chromatography

    Analysis of G . morsitans salivary glycoproteins. 10 μg of G . morsitans salivary proteins (lanes 1 and 2) and 10 μg of egg albumin (lanes 3 and 4) were incubated overnight with (2 and 4) and without (1 and 3) PNGase F. After digestion, proteins were resolved by SDS-PAGE and Coomassie blue-stained. There was a notable shift in migration in 4 bands following PNGase F treatment. After in-gel trypsinization and MALDI-TOF MS analysis these bands were identified as 5’ Nucleotidase (1), TSGF 2/Adenosine deaminase (2), TSGF 1 (3), and Tsal 1/2 (4). *, PNGase F enzyme.
    Figure Legend Snippet: Analysis of G . morsitans salivary glycoproteins. 10 μg of G . morsitans salivary proteins (lanes 1 and 2) and 10 μg of egg albumin (lanes 3 and 4) were incubated overnight with (2 and 4) and without (1 and 3) PNGase F. After digestion, proteins were resolved by SDS-PAGE and Coomassie blue-stained. There was a notable shift in migration in 4 bands following PNGase F treatment. After in-gel trypsinization and MALDI-TOF MS analysis these bands were identified as 5’ Nucleotidase (1), TSGF 2/Adenosine deaminase (2), TSGF 1 (3), and Tsal 1/2 (4). *, PNGase F enzyme.

    Techniques Used: Incubation, SDS Page, Staining, Migration

    Analysis of tsetse salivary  N -linked glycans in teneral, naïve and trypanosome-infected flies. (A) Comparison of HILIC-UHPLC profiles of salivary  N -glycans released by PNGase F. Analysis of 2AB-labelled glycans from (i) teneral, (ii) naïve, and (iii) trypanosome-infected saliva. Relative abundances are indicated in   Table 2 . Tbb,  Trypanosoma brucei brucei . (B) Positive-ion ESI-MS analysis of procainamide labelled  N- glycans from adult naïve and trypanosome-infected saliva. Spectra are shown for naïve (top) and trypanosome-infected (bottom) saliva. Numbers refer to the structures shown in   Table 1 . Green circle, mannose; blue square,  N -Acetylglucosamine; red triangle, fucose; Proc, procainamide. Peaks labelled with an asterisk refer to buffer contaminants.
    Figure Legend Snippet: Analysis of tsetse salivary N -linked glycans in teneral, naïve and trypanosome-infected flies. (A) Comparison of HILIC-UHPLC profiles of salivary N -glycans released by PNGase F. Analysis of 2AB-labelled glycans from (i) teneral, (ii) naïve, and (iii) trypanosome-infected saliva. Relative abundances are indicated in Table 2 . Tbb, Trypanosoma brucei brucei . (B) Positive-ion ESI-MS analysis of procainamide labelled N- glycans from adult naïve and trypanosome-infected saliva. Spectra are shown for naïve (top) and trypanosome-infected (bottom) saliva. Numbers refer to the structures shown in Table 1 . Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide. Peaks labelled with an asterisk refer to buffer contaminants.

    Techniques Used: Infection, Hydrophilic Interaction Liquid Chromatography

    24) Product Images from "Protection against influenza infection requires early recognition by inflammatory dendritic cells through C-type lectin receptor SIGN-R1"

    Article Title: Protection against influenza infection requires early recognition by inflammatory dendritic cells through C-type lectin receptor SIGN-R1

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0506-6

    IDC express the lectin receptor SIGN-R1 that recognises influenza PR8 through binding to glycosylated viral proteins. a , Flow cytometric analysis showing the expression levels of SIGN-R1 in tracheal DC and RTMϕ at day 3 p.i. (n = 5 mice per group).  b , Representative scatterplots showing the percentage of SIGN-R1+ IDC in trachea at day 3 p.i..  c , Representative 3D reconstruction of confocal micrographs showing the colocalisation between DIO-labelled PR8 (PR8-DIO) and SIGN-R1 (white arrows), expressed in an IDC one hour after  in vitro  culture. Bar indicates 2 μm (left) and 0.5 μm (right).  d , Sensogramms of Surface Plasmon Resonance (SPR) analysis of the HA-binding affinity of SIGN-R1 Fc chimera protein (SIGN-R1-Fc.) SIGN-R1-Fc with or without glycosylation (after treatment with N-glycosidase F (PNGase F)), with different concentration of H1, below their respective kinetic parameters, calculated by SPR using purified proteins are shown. N.B., no binding detected.  e , ELISA analysis of the HA-binding titre from different subtypes of the recombinant mouse SIGN-R1-Fc (n = 2 replicates per group).  f , (Left) Representative scatterplots from flow cytometric analysis showing the capture of PR8-DIO by SIGN-R1+ IDC after an incubation period of 1 h with the virus. In the αSIGN-R1 group, the binding was inhibited by administration of a blocking antibody previous to the incubation with the virus. (Right) Mean fluorescence intensity (MFI) analysis showing differences in the capture of PR8-DIO by IDC treated with αSIGN-R1 blocking antibody with respect to the control groups (n = 4, 4 and 3 replicates per respective group). The presented data are representative of at least three ( a-c  and  f ) or two ( d-e ) independent experiments. Results are given as mean ± SD ( a ,  b  and  f ), as mean± SEM ( d ) or mean alone ( e ).
    Figure Legend Snippet: IDC express the lectin receptor SIGN-R1 that recognises influenza PR8 through binding to glycosylated viral proteins. a , Flow cytometric analysis showing the expression levels of SIGN-R1 in tracheal DC and RTMϕ at day 3 p.i. (n = 5 mice per group). b , Representative scatterplots showing the percentage of SIGN-R1+ IDC in trachea at day 3 p.i.. c , Representative 3D reconstruction of confocal micrographs showing the colocalisation between DIO-labelled PR8 (PR8-DIO) and SIGN-R1 (white arrows), expressed in an IDC one hour after in vitro culture. Bar indicates 2 μm (left) and 0.5 μm (right). d , Sensogramms of Surface Plasmon Resonance (SPR) analysis of the HA-binding affinity of SIGN-R1 Fc chimera protein (SIGN-R1-Fc.) SIGN-R1-Fc with or without glycosylation (after treatment with N-glycosidase F (PNGase F)), with different concentration of H1, below their respective kinetic parameters, calculated by SPR using purified proteins are shown. N.B., no binding detected. e , ELISA analysis of the HA-binding titre from different subtypes of the recombinant mouse SIGN-R1-Fc (n = 2 replicates per group). f , (Left) Representative scatterplots from flow cytometric analysis showing the capture of PR8-DIO by SIGN-R1+ IDC after an incubation period of 1 h with the virus. In the αSIGN-R1 group, the binding was inhibited by administration of a blocking antibody previous to the incubation with the virus. (Right) Mean fluorescence intensity (MFI) analysis showing differences in the capture of PR8-DIO by IDC treated with αSIGN-R1 blocking antibody with respect to the control groups (n = 4, 4 and 3 replicates per respective group). The presented data are representative of at least three ( a-c and f ) or two ( d-e ) independent experiments. Results are given as mean ± SD ( a , b and f ), as mean± SEM ( d ) or mean alone ( e ).

    Techniques Used: Binding Assay, Flow Cytometry, Expressing, Mouse Assay, In Vitro, SPR Assay, Concentration Assay, Purification, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Blocking Assay, Fluorescence

    25) Product Images from "Semaphorin-7A Is an Erythrocyte Receptor for P. falciparum Merozoite-Specific TRAP Homolog, MTRAP"

    Article Title: Semaphorin-7A Is an Erythrocyte Receptor for P. falciparum Merozoite-Specific TRAP Homolog, MTRAP

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003031

    The MTRAP-Semaphorin-7A interaction is not influenced by glycans. ( A ) PNGase F treatment of Semaphorin-7A. Biotinylated Semaphorin-7A was incubated with PNGase F for 10, 30 or 60 minutes. Enzyme-treated and untreated Semaphorin-7A were resolved by SDS-PAGE under reducing conditions and detected by Western blotting using Streptavidin-HRP. ( B ) Binding of MTRAP to PNGase F-treated Semaphorin-7A was indistinguishable from untreated Semaphorin-7A using the AVEXIS assay. MTRAP was used as the prey against Semaphorin-7A baits. (+) = positive control, (−) = negative control. Bar graphs show mean ± SEM, n = 3. ( C ) PNGase F treatment did not quantitatively influence MTRAP binding to Semaphorin-7A using SPR. Three concentrations of purified monomeric MTRAP were injected over flow cells immobilised with PNGase F-treated and untreated Semaphorin-7A. Dissociation rate constants ( k d ) were calculated to be 0.063±0.00007 s −1 for PNGase F treated, and 0.061±0.00006 s −1 for untreated Semaphorin-7A, by fitting a first order dissociation model to the washout phase of the binding curves. Shown are the normalized, averaged values ± SEM, n = 3. ( D ) MTRAP does not interact with sulphated glycoconjugates. Purified monomeric Semaphorin-7A, chondroitin sulphate A, chondroitin sulphate C, dextran sulphate, heparin and heparan sulphate were injected at 1 mg/ml over MTRAP immobilised on a streptavidin-coated sensor chip.
    Figure Legend Snippet: The MTRAP-Semaphorin-7A interaction is not influenced by glycans. ( A ) PNGase F treatment of Semaphorin-7A. Biotinylated Semaphorin-7A was incubated with PNGase F for 10, 30 or 60 minutes. Enzyme-treated and untreated Semaphorin-7A were resolved by SDS-PAGE under reducing conditions and detected by Western blotting using Streptavidin-HRP. ( B ) Binding of MTRAP to PNGase F-treated Semaphorin-7A was indistinguishable from untreated Semaphorin-7A using the AVEXIS assay. MTRAP was used as the prey against Semaphorin-7A baits. (+) = positive control, (−) = negative control. Bar graphs show mean ± SEM, n = 3. ( C ) PNGase F treatment did not quantitatively influence MTRAP binding to Semaphorin-7A using SPR. Three concentrations of purified monomeric MTRAP were injected over flow cells immobilised with PNGase F-treated and untreated Semaphorin-7A. Dissociation rate constants ( k d ) were calculated to be 0.063±0.00007 s −1 for PNGase F treated, and 0.061±0.00006 s −1 for untreated Semaphorin-7A, by fitting a first order dissociation model to the washout phase of the binding curves. Shown are the normalized, averaged values ± SEM, n = 3. ( D ) MTRAP does not interact with sulphated glycoconjugates. Purified monomeric Semaphorin-7A, chondroitin sulphate A, chondroitin sulphate C, dextran sulphate, heparin and heparan sulphate were injected at 1 mg/ml over MTRAP immobilised on a streptavidin-coated sensor chip.

    Techniques Used: Incubation, SDS Page, Western Blot, Binding Assay, Positive Control, Negative Control, SPR Assay, Purification, Injection, Flow Cytometry, Chromatin Immunoprecipitation

    26) Product Images from "SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors"

    Article Title: SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.227462

    Binding of CLRs to the recombinant SARS-CoV-2 S. ( a-e ) Immunoblots with human Fc-fused CLRs DC-SIGN ( a ), L-SIGN ( b ), MR ( c ), Dectin-2 ( d ) and MGL ( e ) to detect recombinant S1 and S after mock enzymatic digestion or with Endo H, PNGase F or Neu digestion. As negative controls, these glycosidases were also included in some assays. EBY-100 represents the lysates of yeast strain EBY-100. BSM is the recombinant bovine submaxillary mucin. In all assays 5 mM Ca 2+ was included in solutions of CLRs. ( f ) Schematic presentation of the cleavage sites of Endo H, PNGase F and Neu on N- and O-glycans. Endo H cleaves the oligomannose and hybrid N-glycans, while PNGase F removes all N-glycans including the complex type. Neu removes all sialic acids on N- or O-glycans. ( g-k ) Affinity constant measurement for DC-SIGN ( g ), L-SIGN ( h ), MR ( i ), MGL ( j ) and ACE2 ( k ) by ELISA assay. The plates were coated by recombinant SARS-CoV-2 S trimer. Error bars represent SD of two replicates. The data were plotted as % binding relative to the saturated binding as 100%. In all assays 5 mM Ca 2+ and Tween-20 were included in solutions of CLRs.
    Figure Legend Snippet: Binding of CLRs to the recombinant SARS-CoV-2 S. ( a-e ) Immunoblots with human Fc-fused CLRs DC-SIGN ( a ), L-SIGN ( b ), MR ( c ), Dectin-2 ( d ) and MGL ( e ) to detect recombinant S1 and S after mock enzymatic digestion or with Endo H, PNGase F or Neu digestion. As negative controls, these glycosidases were also included in some assays. EBY-100 represents the lysates of yeast strain EBY-100. BSM is the recombinant bovine submaxillary mucin. In all assays 5 mM Ca 2+ was included in solutions of CLRs. ( f ) Schematic presentation of the cleavage sites of Endo H, PNGase F and Neu on N- and O-glycans. Endo H cleaves the oligomannose and hybrid N-glycans, while PNGase F removes all N-glycans including the complex type. Neu removes all sialic acids on N- or O-glycans. ( g-k ) Affinity constant measurement for DC-SIGN ( g ), L-SIGN ( h ), MR ( i ), MGL ( j ) and ACE2 ( k ) by ELISA assay. The plates were coated by recombinant SARS-CoV-2 S trimer. Error bars represent SD of two replicates. The data were plotted as % binding relative to the saturated binding as 100%. In all assays 5 mM Ca 2+ and Tween-20 were included in solutions of CLRs.

    Techniques Used: Binding Assay, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay

    27) Product Images from "The GARP Domain of the Rod CNG Channel’s β1-subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disc Rim"

    Article Title: The GARP Domain of the Rod CNG Channel’s β1-subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disc Rim

    Journal: bioRxiv

    doi: 10.1101/2020.10.01.322859

    The rod CNG channel is trafficked using the conventional secretory pathway. Mouse retinal lysates (untreated) were incubated with PNGase F or Endo H and analyzed by Western blotting. ( A ) Electrophoretic mobility of non-glycosylated proteins CNGβ1, guanylate cyclase-2 (GC-2) and Rom-1, is unaffected by enzymatic treatments. ( B ) CNGα1, guanylate cyclase-1 (GC-1) and rhodopsin (Rho) are sensitive to PNGase F and resistant to Endo H treatment, indicating their processing through the conventional secretory pathway. ( C ) ABCA4 and peripherin-2 (Prph2) are sensitive to both PNGase F and Endo H treatments, indicating their processing through the unconventional secretory pathway. Dashed red lines are used to mark the positions of untreated protein bands. 10 µg of total protein was loaded in each lane.
    Figure Legend Snippet: The rod CNG channel is trafficked using the conventional secretory pathway. Mouse retinal lysates (untreated) were incubated with PNGase F or Endo H and analyzed by Western blotting. ( A ) Electrophoretic mobility of non-glycosylated proteins CNGβ1, guanylate cyclase-2 (GC-2) and Rom-1, is unaffected by enzymatic treatments. ( B ) CNGα1, guanylate cyclase-1 (GC-1) and rhodopsin (Rho) are sensitive to PNGase F and resistant to Endo H treatment, indicating their processing through the conventional secretory pathway. ( C ) ABCA4 and peripherin-2 (Prph2) are sensitive to both PNGase F and Endo H treatments, indicating their processing through the unconventional secretory pathway. Dashed red lines are used to mark the positions of untreated protein bands. 10 µg of total protein was loaded in each lane.

    Techniques Used: Incubation, Western Blot

    28) Product Images from "Epistastic Interactions within the Junín Virus Envelope Glycoprotein Complex Provide an Evolutionary Barrier to Reversion in the Live-Attenuated Candid#1 Vaccine"

    Article Title: Epistastic Interactions within the Junín Virus Envelope Glycoprotein Complex Provide an Evolutionary Barrier to Reversion in the Live-Attenuated Candid#1 Vaccine

    Journal: Journal of Virology

    doi: 10.1128/JVI.01682-17

    Effect of A168T and F427I mutations on expression and cell-cell fusion activity of ectopically expressed GPC. (A and B) Metabolically labeled Candid#1 and MC2 GPCs were immunoprecipitated from transfected Vero-76 cells using the anti-GP1 MAb BF11 and resolved using NuPAGE. The mature GP1 and GP2 subunits comigrate as glycoproteins (A) and are resolved following deglycosylation by treatment with PNGase F (B). The uncleaved GP1GP2 precursor is labeled, and deglycosylated polypeptides are indicated as pp. GPC expression was quantitated by using a Fuji FLA3000G phosphorimager and ImageGauge software. Photostimulated luminescence (PSL) values in the respective GP1GP2 precursor bands in panel A are 427,881, 174,626, 360,045, 68,087, 339,735, and 53,852. This pattern of expression was confirmed in three replicate analyses. (C) Cell-cell fusion activity of the ectopically expressed MC2 (red) and Candid#1 (blue) GPCs was triggered by exposure to medium adjusted to pH 5.0 and determined using a chemiluminescent β-galactosidase fusion reporter. Relative light units are plotted. Error bars represent the standard errors of the means from replicate fusion reactions. Complete and partial replicates of this experiment ( n > 4) yielded concordant results. A pairwise statistical comparison using a 2-tailed Mann-Whitney analysis, as implemented in GraphPad Prism software, demonstrated that Can GPC is significantly more fusogenic than MC2 GPC ( P = 0.0002).
    Figure Legend Snippet: Effect of A168T and F427I mutations on expression and cell-cell fusion activity of ectopically expressed GPC. (A and B) Metabolically labeled Candid#1 and MC2 GPCs were immunoprecipitated from transfected Vero-76 cells using the anti-GP1 MAb BF11 and resolved using NuPAGE. The mature GP1 and GP2 subunits comigrate as glycoproteins (A) and are resolved following deglycosylation by treatment with PNGase F (B). The uncleaved GP1GP2 precursor is labeled, and deglycosylated polypeptides are indicated as pp. GPC expression was quantitated by using a Fuji FLA3000G phosphorimager and ImageGauge software. Photostimulated luminescence (PSL) values in the respective GP1GP2 precursor bands in panel A are 427,881, 174,626, 360,045, 68,087, 339,735, and 53,852. This pattern of expression was confirmed in three replicate analyses. (C) Cell-cell fusion activity of the ectopically expressed MC2 (red) and Candid#1 (blue) GPCs was triggered by exposure to medium adjusted to pH 5.0 and determined using a chemiluminescent β-galactosidase fusion reporter. Relative light units are plotted. Error bars represent the standard errors of the means from replicate fusion reactions. Complete and partial replicates of this experiment ( n > 4) yielded concordant results. A pairwise statistical comparison using a 2-tailed Mann-Whitney analysis, as implemented in GraphPad Prism software, demonstrated that Can GPC is significantly more fusogenic than MC2 GPC ( P = 0.0002).

    Techniques Used: Expressing, Activity Assay, Gel Permeation Chromatography, Metabolic Labelling, Labeling, Immunoprecipitation, Transfection, Software, MANN-WHITNEY

    29) Product Images from "The mannosylated extracellular domain of Her2/neu produced in P. pastoris induces protective antitumor immunity"

    Article Title: The mannosylated extracellular domain of Her2/neu produced in P. pastoris induces protective antitumor immunity

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-386

    Deglycosylation of ECD by peptide N-glycosidase F (PNGase F) . Purified ECD incubated for 1 h at 37°C with (+) or without (-) PNGase F was analyzed in 8% SDS-PAGE. HMW, LMW: high and low molecular weight markers.
    Figure Legend Snippet: Deglycosylation of ECD by peptide N-glycosidase F (PNGase F) . Purified ECD incubated for 1 h at 37°C with (+) or without (-) PNGase F was analyzed in 8% SDS-PAGE. HMW, LMW: high and low molecular weight markers.

    Techniques Used: Purification, Incubation, SDS Page, Molecular Weight

    30) Product Images from "Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris"

    Article Title: Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris

    Journal: Osong Public Health and Research Perspectives

    doi: 10.1016/j.phrp.2014.08.005

    Analysis of glycosylation of tick-borne encephalitis virus E proteins in  Pichia pastoris  transformed with plasmid pGAPZɑA/93prM-E. Samples from (A) the cell lysate and (B) the cell supernatant were treated with Endo H (+) or PNGase F (+) and compared with untreated controls (−) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.   = E protein;   = deglycosylated E protein; Endo H = endoglycosidase H; PNGase =  N -glycosidase F.
    Figure Legend Snippet: Analysis of glycosylation of tick-borne encephalitis virus E proteins in Pichia pastoris transformed with plasmid pGAPZɑA/93prM-E. Samples from (A) the cell lysate and (B) the cell supernatant were treated with Endo H (+) or PNGase F (+) and compared with untreated controls (−) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.  = E protein;  = deglycosylated E protein; Endo H = endoglycosidase H; PNGase =  N -glycosidase F.

    Techniques Used: Transformation Assay, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Western Blot

    31) Product Images from "Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases"

    Article Title: Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases

    Journal: Glycobiology

    doi: 10.1093/glycob/cwu132

    SDS–PAGE analysis of purified, recombinant hexosaminidases. Purified recombinant enzymes were either incubated with water (−) or  N -glycosidase F (+) and subjected to SDS–PAGE and staining with Coomassie Brilliant Blue. Dagger indicates the enzyme produced in  P. pastoris  X-33 and asterisk indicates the enzyme produced in Hi5 insect cells. Band at approximately 30 kDa in (+) lanes corresponds to the  N -glycosidase F protein. All  C. elegans  enzymes were produced in  P. pastoris  X-33.
    Figure Legend Snippet: SDS–PAGE analysis of purified, recombinant hexosaminidases. Purified recombinant enzymes were either incubated with water (−) or N -glycosidase F (+) and subjected to SDS–PAGE and staining with Coomassie Brilliant Blue. Dagger indicates the enzyme produced in P. pastoris X-33 and asterisk indicates the enzyme produced in Hi5 insect cells. Band at approximately 30 kDa in (+) lanes corresponds to the N -glycosidase F protein. All C. elegans enzymes were produced in P. pastoris X-33.

    Techniques Used: SDS Page, Purification, Recombinant, Incubation, Staining, Produced

    32) Product Images from "In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus"

    Article Title: In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183589

    SDS-PAGE analysis of purified plant produced Pfs48/45 variants. Lanes were loaded with ~1.0 μg (A) or ~2.0 μg (B) per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced Pfs48/45proteins. (A) Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. (B) Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C,co-expressed with PNGase F; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with PNGase F. Indications of gPfs48/45, dPfs48/45, gPfs48/45-10C and dPfs48/45-10C are the same as shown in Fig 5 . M: color prestained protein standard (New England Biolabs). Arrow in B (indicating lane:2) shows aggregation of deglycosylated Pfs48/45-10C protein, produced by in vivo deglycosylation of Pfs48/45 co-expressed with PNGase F.
    Figure Legend Snippet: SDS-PAGE analysis of purified plant produced Pfs48/45 variants. Lanes were loaded with ~1.0 μg (A) or ~2.0 μg (B) per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced Pfs48/45proteins. (A) Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. (B) Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C,co-expressed with PNGase F; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with PNGase F. Indications of gPfs48/45, dPfs48/45, gPfs48/45-10C and dPfs48/45-10C are the same as shown in Fig 5 . M: color prestained protein standard (New England Biolabs). Arrow in B (indicating lane:2) shows aggregation of deglycosylated Pfs48/45-10C protein, produced by in vivo deglycosylation of Pfs48/45 co-expressed with PNGase F.

    Techniques Used: SDS Page, Purification, Produced, In Vivo

    SDS-PAGE analysis of plant produced, purified bacterial Endo H from  N .  benthamiana  plants and evaluation of its deglycosylating activity  in vitro . (A) SDS-PAGE analysis of purified plant produced Endo H from  N .  benthamiana  plant. Lanes: 1-About 20 μg of the crude supernatant was loaded; 2–0.75 μg purified Endo H was loaded. (B), (C) Western blot or SDS-PAGE analysis of PA83 protein incubated with either plant produced Endo H or commercial Endo H or commercial PNGase F. Lanes: 1- plant produced PA83; 2- plant produced PA83 was treated with the plant produced Endo H; 3- plant produced PA83 was treated with the commercial Endo H; 4- plant produced PA83 was treated with the commercial PNGase F. 100 ng or 2 μg PA83 protein samples were loaded in each lane in Western blot and SDS-PAGE, respectively. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Arrows in C indicates migration of commercial Endo H and PNGase F.
    Figure Legend Snippet: SDS-PAGE analysis of plant produced, purified bacterial Endo H from N . benthamiana plants and evaluation of its deglycosylating activity in vitro . (A) SDS-PAGE analysis of purified plant produced Endo H from N . benthamiana plant. Lanes: 1-About 20 μg of the crude supernatant was loaded; 2–0.75 μg purified Endo H was loaded. (B), (C) Western blot or SDS-PAGE analysis of PA83 protein incubated with either plant produced Endo H or commercial Endo H or commercial PNGase F. Lanes: 1- plant produced PA83; 2- plant produced PA83 was treated with the plant produced Endo H; 3- plant produced PA83 was treated with the commercial Endo H; 4- plant produced PA83 was treated with the commercial PNGase F. 100 ng or 2 μg PA83 protein samples were loaded in each lane in Western blot and SDS-PAGE, respectively. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Arrows in C indicates migration of commercial Endo H and PNGase F.

    Techniques Used: SDS Page, Produced, Purification, Activity Assay, In Vitro, Western Blot, Incubation, Migration

    Schematic representation of Endo H or PNGase F cleavages. (A) Endo H cleaves between the two GlcNAc residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one GlcNAc remaining on the asparagines (Asn). (B) Peptide - N -Glycosidase F (PNGase F), is an amidase that cleaves the bond between GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N -linked glycoproteins;
    Figure Legend Snippet: Schematic representation of Endo H or PNGase F cleavages. (A) Endo H cleaves between the two GlcNAc residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one GlcNAc remaining on the asparagines (Asn). (B) Peptide - N -Glycosidase F (PNGase F), is an amidase that cleaves the bond between GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N -linked glycoproteins;

    Techniques Used:

    Study of the deglycosylation efficiency of plant produced Endo-H against PNGase F in vitro . (A) SDS-PAGE analysis of purified plant produced PNGase F and Endo H. Lanes were loaded with 1.0 μg per lane. 1-plant produced PNGase F; 2- plant produced Endo H; M-color prestained protein standard (New England Biolabs). (B) Plant produced PA83 was incubated with different amounts (0, 25, 100, 200, 400, 800 ng) of plant produced Endo H or PNGase F, as indicated. After incubation, proteins were analyzed by SDS-PAGE followed by Western blot analysis. Proteins were detected using a mixture of anti-His Tag antibody to detect His tagged PA83 and anti-FLAG antibody to detect FLAG-tagged Endo H or PNGase F. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). (C) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial Endo H, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M-color prestained protein standard (New England Biolabs). (D) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial PNGase F, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M- color prestained protein standard (New England Biolabs).
    Figure Legend Snippet: Study of the deglycosylation efficiency of plant produced Endo-H against PNGase F in vitro . (A) SDS-PAGE analysis of purified plant produced PNGase F and Endo H. Lanes were loaded with 1.0 μg per lane. 1-plant produced PNGase F; 2- plant produced Endo H; M-color prestained protein standard (New England Biolabs). (B) Plant produced PA83 was incubated with different amounts (0, 25, 100, 200, 400, 800 ng) of plant produced Endo H or PNGase F, as indicated. After incubation, proteins were analyzed by SDS-PAGE followed by Western blot analysis. Proteins were detected using a mixture of anti-His Tag antibody to detect His tagged PA83 and anti-FLAG antibody to detect FLAG-tagged Endo H or PNGase F. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). (C) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial Endo H, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M-color prestained protein standard (New England Biolabs). (D) Plant produced PA83 was incubated at 37°C for 1 h with different amounts (0, 25, 50, 400 and 800 ngs) of commercial PNGase F, as indicated. Lanes, C- PA83 protein was kept at 4°C for 1 h; M- color prestained protein standard (New England Biolabs).

    Techniques Used: Produced, In Vitro, SDS Page, Purification, Incubation, Western Blot, Next-Generation Sequencing

    Glycan detection and Western blot analysis of glycosylated and  in vivo  deglycosylated PA83 and Pfs48/45-10C variants. (A), (C) 0.25 μg of protein from each sample was run on a 10% SDS-PAGE followed by in-gel glycan detection using the Pro-Q Emerald 300 glycoprotein staining kit. Stained proteins were visualized by UV illumination. (B), (D) Western blot analysis of the same samples using anti-His Tag antibody (BioLegend). (A), (B) Lanes: 1 –plant produced glycosylated PA83; 2– deglycosylated PA83, produced by  in vivo  deglycosylation of PA83, co-expressed with Endo H; 3– deglycosylated PA83, produced by  in vivo  deglycosylation of PA83, co-expressed with PNGase F. (C), (D) Lines: 1 –plant produced glycosylated Pfs48/45-10C; 2–deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45-10C, co-expressed with Endo H; 3– deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45-10C, co-expressed with Endo H. M1: CandyCane glycoprotein molecular weight standards (Molecular Probes), 250 ng of each protein per lane. M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Indications of gPA83, dPA83, gPfs48/45-10C, dPfs48/45-10C are the same as shown in   Fig 4 .
    Figure Legend Snippet: Glycan detection and Western blot analysis of glycosylated and in vivo deglycosylated PA83 and Pfs48/45-10C variants. (A), (C) 0.25 μg of protein from each sample was run on a 10% SDS-PAGE followed by in-gel glycan detection using the Pro-Q Emerald 300 glycoprotein staining kit. Stained proteins were visualized by UV illumination. (B), (D) Western blot analysis of the same samples using anti-His Tag antibody (BioLegend). (A), (B) Lanes: 1 –plant produced glycosylated PA83; 2– deglycosylated PA83, produced by in vivo deglycosylation of PA83, co-expressed with Endo H; 3– deglycosylated PA83, produced by in vivo deglycosylation of PA83, co-expressed with PNGase F. (C), (D) Lines: 1 –plant produced glycosylated Pfs48/45-10C; 2–deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with Endo H; 3– deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45-10C, co-expressed with Endo H. M1: CandyCane glycoprotein molecular weight standards (Molecular Probes), 250 ng of each protein per lane. M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Indications of gPA83, dPA83, gPfs48/45-10C, dPfs48/45-10C are the same as shown in Fig 4 .

    Techniques Used: Western Blot, In Vivo, SDS Page, Staining, Produced, Molecular Weight

    Study of stability of glycosylated and deglycosylated PA83 variants using SDS-PAGE analysis. Plant produced, glycosylated PA83, and in vivo Endo H or PNGase F deglycosylated forms of PA83 were purified as described in Materials and Methods. (A) The purified plant produced PA83 variants were stored for 1 hour at 37°C or for 72 hours at 4°C and analyzed by SDS-PAGE. Lanes were loaded with ~8.0 μg per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced PA83 proteins. (B) Purified, plant produced, glycosylated PA83, and in vivo deglycosylated (co-expressed with Endo H or PNGase F) and in vitro deglycosylated (by commercial Endo H) proteins were incubated at 37°C for 1, 4, 8, 16 and 24 hours, and analyzed in SDS-PAGE. Lanes were loaded with ~5.0 μg per lane for each sample. M- color prestained protein standard (New England Biolabs).
    Figure Legend Snippet: Study of stability of glycosylated and deglycosylated PA83 variants using SDS-PAGE analysis. Plant produced, glycosylated PA83, and in vivo Endo H or PNGase F deglycosylated forms of PA83 were purified as described in Materials and Methods. (A) The purified plant produced PA83 variants were stored for 1 hour at 37°C or for 72 hours at 4°C and analyzed by SDS-PAGE. Lanes were loaded with ~8.0 μg per lane for glycosylated, Endo H or PNGase F in vivo deglycosylated plant produced PA83 proteins. (B) Purified, plant produced, glycosylated PA83, and in vivo deglycosylated (co-expressed with Endo H or PNGase F) and in vitro deglycosylated (by commercial Endo H) proteins were incubated at 37°C for 1, 4, 8, 16 and 24 hours, and analyzed in SDS-PAGE. Lanes were loaded with ~5.0 μg per lane for each sample. M- color prestained protein standard (New England Biolabs).

    Techniques Used: SDS Page, Produced, In Vivo, Purification, In Vitro, Incubation

    Western blot analysis of Pfs45/48-10C variants using MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody compared with the anti-His tag antibody. (A) Native PAGE followed by Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-His tag antibody. Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45-10C, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a His Tag antibody.
    Figure Legend Snippet: Western blot analysis of Pfs45/48-10C variants using MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody compared with the anti-His tag antibody. (A) Native PAGE followed by Western blot analysis of Pfs45/48-10C variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-His tag antibody. Lanes: 1- glycosylated Pfs48/45-10C; 2- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45-10C, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a His Tag antibody.

    Techniques Used: Western Blot, Clear Native PAGE, SDS Page, Produced, In Vivo, Recombinant

    Western blot analysis of bacterial Endo H or PNGase F produced in Nicotiana benthamiana plants. N . benthamiana plants were infiltrated with pBI-Endo H or pBI-PNGase F constructs to produce Endo H or PNGase F. Lanes: 1-crude extract prepared from control plant; 2- crude extract prepared from plant infiltrated with bacterial Endo H (pBI-Endo H) and 10, 20 and 40 fold diluted samples were loaded into gel; 3- crude extract prepared from plant infiltrated with bacterial PNGase F (pBI-PNGase F),and 2, 5 or 10 fold diluted samples were loaded into gel; 4- purified plant produced Endo H used as a standard protein; 10 or 25 ng were loaded into gel. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific).
    Figure Legend Snippet: Western blot analysis of bacterial Endo H or PNGase F produced in Nicotiana benthamiana plants. N . benthamiana plants were infiltrated with pBI-Endo H or pBI-PNGase F constructs to produce Endo H or PNGase F. Lanes: 1-crude extract prepared from control plant; 2- crude extract prepared from plant infiltrated with bacterial Endo H (pBI-Endo H) and 10, 20 and 40 fold diluted samples were loaded into gel; 3- crude extract prepared from plant infiltrated with bacterial PNGase F (pBI-PNGase F),and 2, 5 or 10 fold diluted samples were loaded into gel; 4- purified plant produced Endo H used as a standard protein; 10 or 25 ng were loaded into gel. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific).

    Techniques Used: Western Blot, Produced, Construct, Purification

    Western blot analysis of co-expression Bacillus anthracis PA83 (A), Pfs48/45 (B) and Pfs48/45-10C with bacterial Endo H or PNGase F in N . benthamiana plants. (A) Western blot analysis of co-expression of PA83. Lanes: 1- N . benthamiana plant was infiltrated with pBI-PA83 construct, for the production of glycosylated PA83, 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pBI-PA83 or pBI-PNGase F/pBI-PA83 constructs, for the production of Endo H (2) or PNGase F (3) deglycosylated PA83 proteins. (B) Western blot analysis of co-expression of Pfs48/45. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45 construct for the production of glycosylated Pfs48/45;2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45 proteins. (C) Western blot analysis of co-expression of Pfs48/45-10C. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45-10C construct for the production of glycosylated Pfs48/45-10C; 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45-10C proteins. gPA83- glycosylated PA83; dPA83- deglycosylated PA83; gPfs48/45: glycosylated Pfs48/45; dPfs48/45: deglycosylated Pfs48/45; gPfs48/45-10C: glycosylated Pfs48/45-10C; dPfs48/45-10C: deglycosylated Pfs48/45-10C.M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). PA83 proteins were detected using the anti-Bacillus anthracis protective antigen antibody BAP0101 (Cat. No. ab1988, Abcam); Ps48/45, Endo H or PNGase F proteins were detected using the anti-FLAG antibody (BioLegend). Pfs48/45-10C protein was detected using the purified anti-His Tag antibody (Cat. No. 652502, BioLegend).
    Figure Legend Snippet: Western blot analysis of co-expression Bacillus anthracis PA83 (A), Pfs48/45 (B) and Pfs48/45-10C with bacterial Endo H or PNGase F in N . benthamiana plants. (A) Western blot analysis of co-expression of PA83. Lanes: 1- N . benthamiana plant was infiltrated with pBI-PA83 construct, for the production of glycosylated PA83, 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pBI-PA83 or pBI-PNGase F/pBI-PA83 constructs, for the production of Endo H (2) or PNGase F (3) deglycosylated PA83 proteins. (B) Western blot analysis of co-expression of Pfs48/45. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45 construct for the production of glycosylated Pfs48/45;2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45 proteins. (C) Western blot analysis of co-expression of Pfs48/45-10C. Lanes: 1- N . benthamiana plant was infiltrated with pEAQ-Pfs48/45-10C construct for the production of glycosylated Pfs48/45-10C; 2,3- N . benthamiana plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45-10C proteins. gPA83- glycosylated PA83; dPA83- deglycosylated PA83; gPfs48/45: glycosylated Pfs48/45; dPfs48/45: deglycosylated Pfs48/45; gPfs48/45-10C: glycosylated Pfs48/45-10C; dPfs48/45-10C: deglycosylated Pfs48/45-10C.M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). PA83 proteins were detected using the anti-Bacillus anthracis protective antigen antibody BAP0101 (Cat. No. ab1988, Abcam); Ps48/45, Endo H or PNGase F proteins were detected using the anti-FLAG antibody (BioLegend). Pfs48/45-10C protein was detected using the purified anti-His Tag antibody (Cat. No. 652502, BioLegend).

    Techniques Used: Western Blot, Expressing, Construct, Purification

    Western blot analysis of Pfs45/48 variants using the MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48 variants using the MRA-26 antibody compared with the anti-FLAG antibody (A) Native PAGE followed by Western blot analysis of Pfs45/48 variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-FLAG antibody. Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by  in vivo  deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a FLAG antibody.
    Figure Legend Snippet: Western blot analysis of Pfs45/48 variants using the MRA-26 antibody, a conformational specific Pfs48/45 mAb. Western blot analysis of Pfs45/48 variants using the MRA-26 antibody compared with the anti-FLAG antibody (A) Native PAGE followed by Western blot analysis of Pfs45/48 variants using the MRA-26 antibody. (B) Samples that were analyzed by Native PAGE, were also analyzed on SDS-PAGE, and proteins were probed with anti-FLAG antibody. Lanes: 1- glycosylated Pfs48/45; 2- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with Endo H; 3- deglycosylated Pfs48/45, produced by in vivo deglycosylation of Pfs48/45, co-expressed with PNGase F. Reduced (R) and non-reduced (N) samples were prepared as described in the Materials and Methods. M1: color prestained protein standard (New England Biolabs); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). Western blot using a conformation-specific anti-Pfs48/45 antibody showed that reduction of the plant produced Pfs48/45 recombinant protein prevents recognition by antibody when compared with Western analysis using a FLAG antibody.

    Techniques Used: Western Blot, Clear Native PAGE, SDS Page, Produced, In Vivo, Recombinant

    Evaluation of the deglycosylation efficiency of Pfs48/45-10C by Endo H or PNGase F in vivo . Pfs48/45-10C (A), Pfs48/45 (B) and PA83 (C) were co-expressed with Endo H or PNGase F at different ratios of OD600 of Agrobacteria carrying Endo H, PNGase F and target genes, as indicated. The efficiency of deglycosylation of target proteins by Endo H or PNGase F was evaluated by Western blot analysis. Size reduction of Pfs48/45-10C, Pfs48/45 and PA83 show as an indicator of glycan removal. Proteins were probed with the anti-4xHis Tag mAb (BioLegend) (A,C) or anti-FLAG antibody (B). Indications of proteins in figures are the same as shown above.
    Figure Legend Snippet: Evaluation of the deglycosylation efficiency of Pfs48/45-10C by Endo H or PNGase F in vivo . Pfs48/45-10C (A), Pfs48/45 (B) and PA83 (C) were co-expressed with Endo H or PNGase F at different ratios of OD600 of Agrobacteria carrying Endo H, PNGase F and target genes, as indicated. The efficiency of deglycosylation of target proteins by Endo H or PNGase F was evaluated by Western blot analysis. Size reduction of Pfs48/45-10C, Pfs48/45 and PA83 show as an indicator of glycan removal. Proteins were probed with the anti-4xHis Tag mAb (BioLegend) (A,C) or anti-FLAG antibody (B). Indications of proteins in figures are the same as shown above.

    Techniques Used: In Vivo, Western Blot

    33) Product Images from "An AA9-LPMO containing a CBM1 domain in Aspergillus nidulans is active on cellulose and cleaves cello-oligosaccharides"

    Article Title: An AA9-LPMO containing a CBM1 domain in Aspergillus nidulans is active on cellulose and cleaves cello-oligosaccharides

    Journal: AMB Express

    doi: 10.1186/s13568-018-0701-5

    Production of AN1602 in P. pastoris and analysis. a Time course of AN1602 secretion over a period of 3 days from P. pastoris cultures after induction with 1.0% methanol. Aliquots of P. pastoris culture media taken at 24 h intervals were assessed in SDS-PAGE gel under denaturing conditions. b SDS-PAGE analysis of purified AN1602. AN1602 protein was treated with PNGase F and loaded onto a denaturing PAGE gel as follows: purified AN1602 (lane1), AN1602 after PNGase F treatment (lane 2), bovine feutin (BF) (lane 3), BF after PNGase F treatment (lane 4), Marker—low molecular protein standard. Control reactions with BF in lane 3 and lane 4 shows the PNGase F enzyme is effective in deglycosylating BF. c Binding of AN1602 to insoluble cellulose. AN1602 protein was mixed with Avicel and incubated on ice for 3 h in sodium phosphate buffer and the PAGE gel was loaded as fractions containing initial starting material (lane 1), supernatant (lane 2), wash (lane 3) and protein bound to pellet (lane 4). Marker—low molecular protein standard. d Sequence features of AN1602. A partial multiple sequence alignment of selected LPMOs. The conserved [Hx n GP] motif containing the second His ligand of the copper active site is highlighted in yellow, and the conserved serine in green. The predicted L3 loop region is marked by a horizontal bar and the first residue of the loop is shown in red. Regioselectivity is (C1/C4) is indicated on the left based on published literature (Frandsen et al. 2016 ; Isaksen et al. 2014 ; Jagadeeswaran et al. 2016 )
    Figure Legend Snippet: Production of AN1602 in P. pastoris and analysis. a Time course of AN1602 secretion over a period of 3 days from P. pastoris cultures after induction with 1.0% methanol. Aliquots of P. pastoris culture media taken at 24 h intervals were assessed in SDS-PAGE gel under denaturing conditions. b SDS-PAGE analysis of purified AN1602. AN1602 protein was treated with PNGase F and loaded onto a denaturing PAGE gel as follows: purified AN1602 (lane1), AN1602 after PNGase F treatment (lane 2), bovine feutin (BF) (lane 3), BF after PNGase F treatment (lane 4), Marker—low molecular protein standard. Control reactions with BF in lane 3 and lane 4 shows the PNGase F enzyme is effective in deglycosylating BF. c Binding of AN1602 to insoluble cellulose. AN1602 protein was mixed with Avicel and incubated on ice for 3 h in sodium phosphate buffer and the PAGE gel was loaded as fractions containing initial starting material (lane 1), supernatant (lane 2), wash (lane 3) and protein bound to pellet (lane 4). Marker—low molecular protein standard. d Sequence features of AN1602. A partial multiple sequence alignment of selected LPMOs. The conserved [Hx n GP] motif containing the second His ligand of the copper active site is highlighted in yellow, and the conserved serine in green. The predicted L3 loop region is marked by a horizontal bar and the first residue of the loop is shown in red. Regioselectivity is (C1/C4) is indicated on the left based on published literature (Frandsen et al. 2016 ; Isaksen et al. 2014 ; Jagadeeswaran et al. 2016 )

    Techniques Used: SDS Page, Purification, Polyacrylamide Gel Electrophoresis, Marker, Binding Assay, Incubation, Sequencing

    34) Product Images from "Chimeric Influenza A Viruses with a Functional Influenza B Virus Neuraminidase or Hemagglutinin"

    Article Title: Chimeric Influenza A Viruses with a Functional Influenza B Virus Neuraminidase or Hemagglutinin

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.17.9116-9123.2003

    Protein gel analysis of influenza A/WSN and recombinant WSN-BHA/ACT-ATM (P6) viruses. Proteins of purified viruses, either untreated (−) or treated with PNGase F (+), were separated by SDS-PAGE on a 12% gel and stained with Coomassie brilliant blue. Positions of untreated (−) and treated (+) proteins are indicated. The band of added PNGase F is shown to the right by an arrow. The positions of the deglycosylated influenza A/WSN HA 1 (Flu A HA 1 ) and WSN-BHA/ACT-ATM (Flu B HA 1 ) viruses are indicated by arrows on the right. HA 0 , HA precursor protein; HA 2 , HA 2 HA subunit.
    Figure Legend Snippet: Protein gel analysis of influenza A/WSN and recombinant WSN-BHA/ACT-ATM (P6) viruses. Proteins of purified viruses, either untreated (−) or treated with PNGase F (+), were separated by SDS-PAGE on a 12% gel and stained with Coomassie brilliant blue. Positions of untreated (−) and treated (+) proteins are indicated. The band of added PNGase F is shown to the right by an arrow. The positions of the deglycosylated influenza A/WSN HA 1 (Flu A HA 1 ) and WSN-BHA/ACT-ATM (Flu B HA 1 ) viruses are indicated by arrows on the right. HA 0 , HA precursor protein; HA 2 , HA 2 HA subunit.

    Techniques Used: Recombinant, Activated Clotting Time Assay, Purification, SDS Page, Staining

    35) Product Images from "Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay"

    Article Title: Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135474

    Glycosylation pattern of swine H1N1 and avian H5N1 rNAs. rNAs were deglycosylated with PNGase F or Endo H and molecular weights of treated and untreated samples were detected by SDS-PAGE followed by Coomassie staining. Data shown are representative of two independent experiments.
    Figure Legend Snippet: Glycosylation pattern of swine H1N1 and avian H5N1 rNAs. rNAs were deglycosylated with PNGase F or Endo H and molecular weights of treated and untreated samples were detected by SDS-PAGE followed by Coomassie staining. Data shown are representative of two independent experiments.

    Techniques Used: SDS Page, Staining

    36) Product Images from "MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation"

    Article Title: MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation

    Journal: mBio

    doi: 10.1128/mBio.01882-20

    MARCH8 blocks the HIV-1 Env and H5N1 HA maturation. (A) FLAG-tagged furin was expressed with MARCH8 and HIV-1 Env or H5N1 HA in 293T cells. Proteins were immunoprecipitated with anti-FLAG and analyzed by WB. (B) HIV-1 Env and H5N1 HA were expressed with MARCH8 in 293T cells, and their processing was determined by WB. (C) HIV-1 Env and H5N1 HA were expressed with MARCH8 in 293T cells. After lysis of cells, cell lysate was treated with Endo H or PNGase F, or left untreated, and analyzed by WB.
    Figure Legend Snippet: MARCH8 blocks the HIV-1 Env and H5N1 HA maturation. (A) FLAG-tagged furin was expressed with MARCH8 and HIV-1 Env or H5N1 HA in 293T cells. Proteins were immunoprecipitated with anti-FLAG and analyzed by WB. (B) HIV-1 Env and H5N1 HA were expressed with MARCH8 in 293T cells, and their processing was determined by WB. (C) HIV-1 Env and H5N1 HA were expressed with MARCH8 in 293T cells. After lysis of cells, cell lysate was treated with Endo H or PNGase F, or left untreated, and analyzed by WB.

    Techniques Used: Immunoprecipitation, Western Blot, Lysis

    MARCH8 blocks the EBOV GP modification by  Nc - and  O -glycans. (A) GP, GPΔFR, GPΔMLD, or GPΔMLDΔFR was expressed with human MARCH8 in 293T cells. After lysis, cell lysate was treated with Endo H or PNGase F, or left untreated, and analyzed by WB. (B) GPΔFR and GPΔMLDΔFR were expressed with EBOV VP40 and human MARCH8 or furin in 293T cells. EBOV VLPs were purified from supernatants via ultracentrifugation. EBOV GP, MARCH8, or furin expressions in cells and/or VLPs were determined by WB.
    Figure Legend Snippet: MARCH8 blocks the EBOV GP modification by Nc - and O -glycans. (A) GP, GPΔFR, GPΔMLD, or GPΔMLDΔFR was expressed with human MARCH8 in 293T cells. After lysis, cell lysate was treated with Endo H or PNGase F, or left untreated, and analyzed by WB. (B) GPΔFR and GPΔMLDΔFR were expressed with EBOV VP40 and human MARCH8 or furin in 293T cells. EBOV VLPs were purified from supernatants via ultracentrifugation. EBOV GP, MARCH8, or furin expressions in cells and/or VLPs were determined by WB.

    Techniques Used: Modification, Lysis, Western Blot, Purification

    37) Product Images from "Dectin-1 Activation on Macrophages by Galectin-9 Promotes Pancreatic Carcinoma and Peritumoral Immune-Tolerance"

    Article Title: Dectin-1 Activation on Macrophages by Galectin-9 Promotes Pancreatic Carcinoma and Peritumoral Immune-Tolerance

    Journal: Nature medicine

    doi: 10.1038/nm.4314

    Galectin-9 is a novel Dectin-1 ligand in PDA (a)  PDA-infiltrating and splenic Gr1 + CD11b +  neutrophils and inflammatory monocytes, CD11c − Gr1 − CD11b + F4/80 +  macrophages, and CD11c + MHCII +  DC from mice harboring orthotopic KPC tumors were gated by flow cytometry and tested for expression of Galectin-9. Gates are based on respective isotype control (not shown). Representative contour plots and quantitative data from 5 mice are shown.  (b)  CD45 − CD133 +  pancreatic cancer cells from orthotopic KPC tumors were gated by flow cytometry and tested for expression of Galectin-9 compared with isotype control. Representative data from  > 3 experiments is shown.  (c)  CD45 +  and CD45 −  cells from human PDA tumor tissue were tested for expression of Galectin-9 compared with PBMC. Representative data from one of three patients are shown.  (d)  Frozen sections of orthotopic KPC-derived pancreatic tumors were co-stained for CD45 and Galectin-9 or isotype control and imaged by confocal microscopy. Representative images are shown (scale bar = 50μm).  (e)  Frozen sections of orthotopic KPC-derived pancreatic tumors were co-stained for CK19 and Galectin-9 or isotype control and imaged by confocal microscopy. Representative images are shown (scale bar = 50μm).  (f)  Protein G-magnetic beads were loaded with the Dectin-1 IgG Fc fusion protein. After blocking, the bead–IgG Fc complexes were incubated with recombinant Galectin-9 and then stained with fluorescently-conjugated anti-Galectin-9 and tested for fluorescence by flow cytometry. Controls included: unstained beads, bead–IgG Fc complexes + recombinant Galectin-9 + fluorescently-conjugated isotype antibody, bead–IgG Fc complexes + fluorescently-conjugated anti-Galectin-9, beads without Dectin-1 IgG Fc incubated with recombinant Galectin-9 + fluorescently-conjugated anti-Galectin-9. To test for competitive inhibition of Galectin-9 binding with a well-characterized Dectin-1 ligand, the bead–IgG Fc complexes were incubated with recombinant Galectin-9 together with d-Zymosan and then stained with fluorescently-conjugated anti-Galectin-9. This assay was repeated twice with similar results.  (g, h)  Galectin-9 coated ELISA plates were incubated with increasing doses of (g) murine or (h) human Dectin-1 IgG Fc or control IgG Fc. The Galectin-9-bound Dectin-1 IgG Fc was detected with anti-IgG-HRP. Averages of triplicates are shown. ELISA assays was repeated twice with similar results.  (i)  Galectin-3, Galectin-4, and Galectin-9 coated ELISA plates were incubated with Dectin-1 IgG Fc or control IgG Fc (2.5μg/ml) in parallel. The Galectin-bound Dectin-1 IgG Fc was detected with anti-IgG-HRP. Averages of triplicates are shown.  (j)  We precipitated Dectin-1 ligands in pancreatic tissue extract from 6 month-old KC mice using the Dectin-1 IgG Fc or control IgG Fc and then probed for Galectin-9 by western blotting. Recombinant Galectin-9 was used as a positive control. This assay was repeated twice with similar results.  (k)  Dectin-1 IgG Fc was treated with either buffer or PNGase F, loaded onto Protein G beads and incubated with recombinant mouse Galectin-9 pre-incubated with 100 mM lactose or buffer. Treated and control samples were analyzed by SDS-PAGE and gels were stained with Coomassie Blue. This experiment was repeated twice with similar results. ( l ) WT and Dectin-1 −/−  macrophages were treated with Galectin-9 (10ug/ml) for 3 hours. Syk phosphorylation was determined by flow cytometry compared with isotype control. Representative histogram overlays and quantitative data from 5 separate experiments are shown.  (m)  Dectin-1 reporter HEK293 cells were untreated or treated low and high doses of Galectin-9 or well-characterized Dectin-1 ligands Curdlan and d-Zymosan. Dectin-1 activation was measured by detection of secreted embryonic alkaline phosphatase. This assay was performed in triplicate (*p
    Figure Legend Snippet: Galectin-9 is a novel Dectin-1 ligand in PDA (a) PDA-infiltrating and splenic Gr1 + CD11b + neutrophils and inflammatory monocytes, CD11c − Gr1 − CD11b + F4/80 + macrophages, and CD11c + MHCII + DC from mice harboring orthotopic KPC tumors were gated by flow cytometry and tested for expression of Galectin-9. Gates are based on respective isotype control (not shown). Representative contour plots and quantitative data from 5 mice are shown. (b) CD45 − CD133 + pancreatic cancer cells from orthotopic KPC tumors were gated by flow cytometry and tested for expression of Galectin-9 compared with isotype control. Representative data from > 3 experiments is shown. (c) CD45 + and CD45 − cells from human PDA tumor tissue were tested for expression of Galectin-9 compared with PBMC. Representative data from one of three patients are shown. (d) Frozen sections of orthotopic KPC-derived pancreatic tumors were co-stained for CD45 and Galectin-9 or isotype control and imaged by confocal microscopy. Representative images are shown (scale bar = 50μm). (e) Frozen sections of orthotopic KPC-derived pancreatic tumors were co-stained for CK19 and Galectin-9 or isotype control and imaged by confocal microscopy. Representative images are shown (scale bar = 50μm). (f) Protein G-magnetic beads were loaded with the Dectin-1 IgG Fc fusion protein. After blocking, the bead–IgG Fc complexes were incubated with recombinant Galectin-9 and then stained with fluorescently-conjugated anti-Galectin-9 and tested for fluorescence by flow cytometry. Controls included: unstained beads, bead–IgG Fc complexes + recombinant Galectin-9 + fluorescently-conjugated isotype antibody, bead–IgG Fc complexes + fluorescently-conjugated anti-Galectin-9, beads without Dectin-1 IgG Fc incubated with recombinant Galectin-9 + fluorescently-conjugated anti-Galectin-9. To test for competitive inhibition of Galectin-9 binding with a well-characterized Dectin-1 ligand, the bead–IgG Fc complexes were incubated with recombinant Galectin-9 together with d-Zymosan and then stained with fluorescently-conjugated anti-Galectin-9. This assay was repeated twice with similar results. (g, h) Galectin-9 coated ELISA plates were incubated with increasing doses of (g) murine or (h) human Dectin-1 IgG Fc or control IgG Fc. The Galectin-9-bound Dectin-1 IgG Fc was detected with anti-IgG-HRP. Averages of triplicates are shown. ELISA assays was repeated twice with similar results. (i) Galectin-3, Galectin-4, and Galectin-9 coated ELISA plates were incubated with Dectin-1 IgG Fc or control IgG Fc (2.5μg/ml) in parallel. The Galectin-bound Dectin-1 IgG Fc was detected with anti-IgG-HRP. Averages of triplicates are shown. (j) We precipitated Dectin-1 ligands in pancreatic tissue extract from 6 month-old KC mice using the Dectin-1 IgG Fc or control IgG Fc and then probed for Galectin-9 by western blotting. Recombinant Galectin-9 was used as a positive control. This assay was repeated twice with similar results. (k) Dectin-1 IgG Fc was treated with either buffer or PNGase F, loaded onto Protein G beads and incubated with recombinant mouse Galectin-9 pre-incubated with 100 mM lactose or buffer. Treated and control samples were analyzed by SDS-PAGE and gels were stained with Coomassie Blue. This experiment was repeated twice with similar results. ( l ) WT and Dectin-1 −/− macrophages were treated with Galectin-9 (10ug/ml) for 3 hours. Syk phosphorylation was determined by flow cytometry compared with isotype control. Representative histogram overlays and quantitative data from 5 separate experiments are shown. (m) Dectin-1 reporter HEK293 cells were untreated or treated low and high doses of Galectin-9 or well-characterized Dectin-1 ligands Curdlan and d-Zymosan. Dectin-1 activation was measured by detection of secreted embryonic alkaline phosphatase. This assay was performed in triplicate (*p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Derivative Assay, Staining, Confocal Microscopy, Magnetic Beads, Blocking Assay, Incubation, Recombinant, Fluorescence, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, SDS Page, Activation Assay

    38) Product Images from "Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris"

    Article Title: Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-2-207

    Deglycosylation of recombinant SOD by treatment with PNGase F . Crude and purified supernatant (SN) samples from the SMD1168H-pGAPZαA-SOD strain were treated without (-) or with (+) 1000 U PNGase F. After the deglycosylation treatment the samples were analyzed by SDS-PAGE and immunoblotting using myc-specific (Myc) antisera. Mw, molecular weight marker (Fermentas).
    Figure Legend Snippet: Deglycosylation of recombinant SOD by treatment with PNGase F . Crude and purified supernatant (SN) samples from the SMD1168H-pGAPZαA-SOD strain were treated without (-) or with (+) 1000 U PNGase F. After the deglycosylation treatment the samples were analyzed by SDS-PAGE and immunoblotting using myc-specific (Myc) antisera. Mw, molecular weight marker (Fermentas).

    Techniques Used: Recombinant, Purification, SDS Page, Molecular Weight, Marker

    39) Product Images from "Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-?B Signaling Pathway"

    Article Title: Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-?B Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075303

    Characterization of CLU-isoform biogenesis. (A) Schematic outline of the 5’-sequence of variant 1 showing the sCLU start codon (framed) and the downstream start codon on exon 3 (underlined). A non-canonical CTG start codon is present on exon 2 (underlined). The SSCR (black shaded nucleotides) and the exon 2/exon 3 border (arrow) are indicated. (B) Western blots of recombinant CLU-V5 proteins in lysates (upper panel) and culture media (lower panel) of HEK-293 cells transiently expressing unmodified or point-mutated (crossed out codons) CLU cDNA variant 1. CLU 34‑449 is translated from the ATG codon on exon 3 (lanes 2, 7). The 50 kDa CLU‑V5 band consists of the sCLU pre-pro-protein (CLU 1‑449 ) translated from the sCLU start codon and CLU 21‑449 translated from the CTG codon (lanes 4, 6). (C) Western blot of recombinant CLU-V5 proteins in lysates of HEK-293 cells transiently expressing sCLU/CLU 1‑449 , CLU 21‑449 or CLU 34‑449 from point-mutated variant 1 cDNAs or unmodified variant 1 cDNA (wildtype). Lysates were treated with PNGase F as indicated. The molecular weights of psCLU and sCLU decrease upon deglycosylation (psCLU/sCLU n.g., lanes 3, 4). PNGase F treatment does not alter the molecular weights of CLU 1‑449 (lanes 3, 4), CLU 21‑449 (lanes 5, 6) and CLU 34‑449 (lanes 7, 8). (D) Western blots of untagged CLU proteins in lysates (upper panel) and culture media (lower panel) of control and MG-132-treated HEK-293 cells transiently expressing sCLU/CLU 1‑449 , CLU 21‑449 or CLU 34‑449 from point-mutated variant 1 cDNAs or transfected with pcDNA (mock). In contrast to CLU 1‑449 and CLU 21‑449 which accumulate upon proteasome inhibition (lanes 3-6), the amount of CLU 34‑449 is not affected (lanes 7, 8). (B, C, D) Data shown are representative of three independent experiments. Lanes are labeled with circled numbers. Recombinant CLU protein bands with a molecular weight of ~38 kDa presumably originate from even further downstream translation initiation sites on CLU cDNAs.
    Figure Legend Snippet: Characterization of CLU-isoform biogenesis. (A) Schematic outline of the 5’-sequence of variant 1 showing the sCLU start codon (framed) and the downstream start codon on exon 3 (underlined). A non-canonical CTG start codon is present on exon 2 (underlined). The SSCR (black shaded nucleotides) and the exon 2/exon 3 border (arrow) are indicated. (B) Western blots of recombinant CLU-V5 proteins in lysates (upper panel) and culture media (lower panel) of HEK-293 cells transiently expressing unmodified or point-mutated (crossed out codons) CLU cDNA variant 1. CLU 34‑449 is translated from the ATG codon on exon 3 (lanes 2, 7). The 50 kDa CLU‑V5 band consists of the sCLU pre-pro-protein (CLU 1‑449 ) translated from the sCLU start codon and CLU 21‑449 translated from the CTG codon (lanes 4, 6). (C) Western blot of recombinant CLU-V5 proteins in lysates of HEK-293 cells transiently expressing sCLU/CLU 1‑449 , CLU 21‑449 or CLU 34‑449 from point-mutated variant 1 cDNAs or unmodified variant 1 cDNA (wildtype). Lysates were treated with PNGase F as indicated. The molecular weights of psCLU and sCLU decrease upon deglycosylation (psCLU/sCLU n.g., lanes 3, 4). PNGase F treatment does not alter the molecular weights of CLU 1‑449 (lanes 3, 4), CLU 21‑449 (lanes 5, 6) and CLU 34‑449 (lanes 7, 8). (D) Western blots of untagged CLU proteins in lysates (upper panel) and culture media (lower panel) of control and MG-132-treated HEK-293 cells transiently expressing sCLU/CLU 1‑449 , CLU 21‑449 or CLU 34‑449 from point-mutated variant 1 cDNAs or transfected with pcDNA (mock). In contrast to CLU 1‑449 and CLU 21‑449 which accumulate upon proteasome inhibition (lanes 3-6), the amount of CLU 34‑449 is not affected (lanes 7, 8). (B, C, D) Data shown are representative of three independent experiments. Lanes are labeled with circled numbers. Recombinant CLU protein bands with a molecular weight of ~38 kDa presumably originate from even further downstream translation initiation sites on CLU cDNAs.

    Techniques Used: Sequencing, Variant Assay, CTG Assay, Western Blot, Recombinant, Expressing, Transfection, Inhibition, Labeling, Molecular Weight

    40) Product Images from "Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin"

    Article Title: Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03151-13

    Recombinant cirsin PSI fused to the α-MF leader sequence is secreted in a unprocessed form. (A) Western blot analysis of culture supernatants of selected transformants for α-MF_PSI(His) 6 and α-MF_PSI(N86S)(His) 6 treated with PNGase F using an anti-His antibody. (B) Western blot analysis of culture supernatant enriched in recombinant wt PSI [α-MF_PSI(His) 6 ] subjected to deglycosylation with endo H using an anti-His antibody. +, presence, and −, absence of glycosidase.
    Figure Legend Snippet: Recombinant cirsin PSI fused to the α-MF leader sequence is secreted in a unprocessed form. (A) Western blot analysis of culture supernatants of selected transformants for α-MF_PSI(His) 6 and α-MF_PSI(N86S)(His) 6 treated with PNGase F using an anti-His antibody. (B) Western blot analysis of culture supernatant enriched in recombinant wt PSI [α-MF_PSI(His) 6 ] subjected to deglycosylation with endo H using an anti-His antibody. +, presence, and −, absence of glycosidase.

    Techniques Used: Recombinant, Sequencing, Western Blot

    The signal peptide in the α-MF domain is sufficient to drive secretion of recombinant cirsin PSI. (A) Schematic representation of SP_PSI(His) 6 (top) and SP_PSI(N86S)_TEV(His) 6 (bottom) fusion proteins. Both constructs contain the signal peptide (SP) of the α-MF domain and a hexahistidine tag. The SP_PSI(His) 6 putative glycosylation site (NET) is indicated; in SP_PSI(N86S)_TEV(His) 6 , this glycosylation site is mutated. The glycosylation mutant also contains a TEV protease cleavage site. (B and C) Analysis of recombinant PSI expression by Western blotting of culture supernatants with an anti-His antibody. (B) SP_PSI(N86S)_TEV(His) 6 . (C) SP_PSI(His) 6 . (D) Protein deglycosylation assays. The culture supernatant of a representative transformant for SP_PSI(His) 6 was treated with PNGase F and endo H and analyzed by Western blotting with an anti-His antibody. +, presence, and −, absence of glycosidase.
    Figure Legend Snippet: The signal peptide in the α-MF domain is sufficient to drive secretion of recombinant cirsin PSI. (A) Schematic representation of SP_PSI(His) 6 (top) and SP_PSI(N86S)_TEV(His) 6 (bottom) fusion proteins. Both constructs contain the signal peptide (SP) of the α-MF domain and a hexahistidine tag. The SP_PSI(His) 6 putative glycosylation site (NET) is indicated; in SP_PSI(N86S)_TEV(His) 6 , this glycosylation site is mutated. The glycosylation mutant also contains a TEV protease cleavage site. (B and C) Analysis of recombinant PSI expression by Western blotting of culture supernatants with an anti-His antibody. (B) SP_PSI(N86S)_TEV(His) 6 . (C) SP_PSI(His) 6 . (D) Protein deglycosylation assays. The culture supernatant of a representative transformant for SP_PSI(His) 6 was treated with PNGase F and endo H and analyzed by Western blotting with an anti-His antibody. +, presence, and −, absence of glycosidase.

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

    Related Articles

    Electrophoresis:

    Article Title: Toward developing recombinant gonadotropin-based hormone therapies for increasing fertility in the flatfish Senegalese sole
    Article Snippet: For this, recombinant gonadotropins (0.5 μg) were diluted in Laemmli sample buffer containing SDS and DTT, denaturated at 95°C, and separated in 12% acrylamide gels. .. The glycosylation state of the proteins was assessed by incubating the samples with 500 units of N-Glycosidase F (PNGase F; New England Biolabs Inc.) for 2 h at 37°C prior to electrophoresis. .. Proteins were blotted onto Immun-Blot® nitrocellulose membranes (Bio-Rad, Spain), blocked in 5% nonfat dry milk in TBST (20 mM Tris, 140 mM NaCl, 0.1% Tween, pH 8) and incubated overnight at 4°C with the Senegalese sole Fshβ and Lhβ antisera diluted 1:5000 in blocking buffer.

    Recombinant:

    Article Title:
    Article Snippet: Approximately 250 μg of protein was produced from 1 liter of yeast culture. .. To determine the extent of LILRA3 glycosylation, the mammalian cell- and yeast-produced recombinant proteins were treated with PNGase F according to the manufacturer's instructions (New England Biolabs). .. In brief, 2 μl of 10× glycoprotein denaturing buffer was added to 2.5 μg of protein in 20 μl of buffer and incubated at 95 °C for 10 min. 3 μl of G7 reaction buffer, 3 μl of Nonidet P-40, and 1 μl (500 units/μl) of PNGase F were then added to each reaction mixture, and samples were incubated at 37 °C for 1 h. Changes in the deglycosylated rLILRA3 size and isoelectric focusing were determined by Western blotting of membranes from one- and two dimensional SDS-PAGE gels, respectively, and mass spectrometry.

    Article Title: Glycosylation of Twisted Gastrulation is Required for BMP Binding and Activity during Craniofacial Development
    Article Snippet: Transfection product proteins, insect cell xTsg, E. coli mTWSG1 and were deglycosylated using PNGase F (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s instructions. .. Recombinant mouse TWSG1 from murine myeloma cells (R & D systems, Minneapolis, MN, USA) was deglycosylated using PNGase F or protein deglycosylation mix (NEB) according to manufacturer’s instructions. .. Blots of FLAG-tagged proteins were probed with 1:1000 Rabbit Anti-DYKDDDDK in 0.1% casein/PBS (Cell Signaling, Danvers, MA, USA).

    Purification:

    Article Title: An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells
    Article Snippet: Deglycosylation of the recombinant HA1 glycoprotein The glycosylation status of the recombinant gHA1 protein was determined with Peptide-N -Glycosidase F (PNGase F) that cleaves the complex oligosaccharides at N-linked glycosylations. .. Briefly, purified gHA1 (3 µg) was denatured in buffer (0.5% SDS and 4 mM DTT), heated at 100°C for 10 min, and subsequently incubated with PNGase F (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. .. The reaction products were resolved by 12% SDS-PAGE, and the presence of HA1 was subsequently determined by immunoblotting, as described above.

    Incubation:

    Article Title: An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells
    Article Snippet: Deglycosylation of the recombinant HA1 glycoprotein The glycosylation status of the recombinant gHA1 protein was determined with Peptide-N -Glycosidase F (PNGase F) that cleaves the complex oligosaccharides at N-linked glycosylations. .. Briefly, purified gHA1 (3 µg) was denatured in buffer (0.5% SDS and 4 mM DTT), heated at 100°C for 10 min, and subsequently incubated with PNGase F (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. .. The reaction products were resolved by 12% SDS-PAGE, and the presence of HA1 was subsequently determined by immunoblotting, as described above.

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    New England Biolabs pngase f
    Purification of the gHA1 from insect cells. (A) SDS-PAGE analysis of AIV HA protein expressed in a baculovirus/insect cell system. His-tagged recombinant hemagglutinin protein (gHA1) was purified using Ni-NTA affinity chromatography and gel filtration. Whole supernatant of insect cell (TriEx Sf9) culture was first loaded onto a Ni-NTA affinity chromatography column (Lane 1). Lanes 2 and 3 are the flow-through and washing eluants through the Ni-NTA affinity column, respectively. Lanes 4 to 6 are collected fractions by imidazole elution. Lane 7 is purified gHA1 after gel filtration chromatography (indicated with an arrow). (B) Cleavage of glycans from the purified gHA1. The purified gHA1 (3 µg) was incubated with (+) or without (–) PNGase F, and the reaction mixture was resolved with 12% SDS-PAGE (left panel). HA was probed with anti-HA polyclonal antibody (right panel). The migration of <t>PNGase</t> F treated (+) and untreated (–) recombinant HA1 is indicated with arrows.
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    Purification of the gHA1 from insect cells. (A) SDS-PAGE analysis of AIV HA protein expressed in a baculovirus/insect cell system. His-tagged recombinant hemagglutinin protein (gHA1) was purified using Ni-NTA affinity chromatography and gel filtration. Whole supernatant of insect cell (TriEx Sf9) culture was first loaded onto a Ni-NTA affinity chromatography column (Lane 1). Lanes 2 and 3 are the flow-through and washing eluants through the Ni-NTA affinity column, respectively. Lanes 4 to 6 are collected fractions by imidazole elution. Lane 7 is purified gHA1 after gel filtration chromatography (indicated with an arrow). (B) Cleavage of glycans from the purified gHA1. The purified gHA1 (3 µg) was incubated with (+) or without (–) PNGase F, and the reaction mixture was resolved with 12% SDS-PAGE (left panel). HA was probed with anti-HA polyclonal antibody (right panel). The migration of PNGase F treated (+) and untreated (–) recombinant HA1 is indicated with arrows.

    Journal: PLoS ONE

    Article Title: An RNA Aptamer That Specifically Binds to the Glycosylated Hemagglutinin of Avian Influenza Virus and Suppresses Viral Infection in Cells

    doi: 10.1371/journal.pone.0097574

    Figure Lengend Snippet: Purification of the gHA1 from insect cells. (A) SDS-PAGE analysis of AIV HA protein expressed in a baculovirus/insect cell system. His-tagged recombinant hemagglutinin protein (gHA1) was purified using Ni-NTA affinity chromatography and gel filtration. Whole supernatant of insect cell (TriEx Sf9) culture was first loaded onto a Ni-NTA affinity chromatography column (Lane 1). Lanes 2 and 3 are the flow-through and washing eluants through the Ni-NTA affinity column, respectively. Lanes 4 to 6 are collected fractions by imidazole elution. Lane 7 is purified gHA1 after gel filtration chromatography (indicated with an arrow). (B) Cleavage of glycans from the purified gHA1. The purified gHA1 (3 µg) was incubated with (+) or without (–) PNGase F, and the reaction mixture was resolved with 12% SDS-PAGE (left panel). HA was probed with anti-HA polyclonal antibody (right panel). The migration of PNGase F treated (+) and untreated (–) recombinant HA1 is indicated with arrows.

    Article Snippet: Briefly, purified gHA1 (3 µg) was denatured in buffer (0.5% SDS and 4 mM DTT), heated at 100°C for 10 min, and subsequently incubated with PNGase F (New England Biolabs, Beverly, MA) according to the manufacturer's protocol.

    Techniques: Purification, SDS Page, Recombinant, Affinity Chromatography, Filtration, Affinity Column, Flow Cytometry, Chromatography, Incubation, Migration

    TWSG1 glycosylation varies depending on host source . mTWSG1 made in murine myeloma cells is markedly glycosylated as indicated by the increase in mobility with PNGase F treatment. Xenopus Tsg made in insect cells is also glycosylated but shows a smaller mobility shift. Murine TWSG1 made in E. coli is not glycosylated and shows no shift in mobility after treatment with PNGase F.

    Journal: Frontiers in Physiology

    Article Title: Glycosylation of Twisted Gastrulation is Required for BMP Binding and Activity during Craniofacial Development

    doi: 10.3389/fphys.2011.00059

    Figure Lengend Snippet: TWSG1 glycosylation varies depending on host source . mTWSG1 made in murine myeloma cells is markedly glycosylated as indicated by the increase in mobility with PNGase F treatment. Xenopus Tsg made in insect cells is also glycosylated but shows a smaller mobility shift. Murine TWSG1 made in E. coli is not glycosylated and shows no shift in mobility after treatment with PNGase F.

    Article Snippet: Recombinant mouse TWSG1 from murine myeloma cells (R & D systems, Minneapolis, MN, USA) was deglycosylated using PNGase F or protein deglycosylation mix (NEB) according to manufacturer’s instructions.

    Techniques: Mobility Shift

    (a) Immunoblots of cauda sperm plasma membrane fraction treated with N-glycanase analyzed by reducing SDS-PAGE on 15% gels and immunostained with anti-TEX101. Lane 1 represents the untreated TEX101 polypeptide, and lane 2 displays N-glycanase treated TEX101 polypeptide. Note the reduction in molecular weight (~17 kDa) of the deglycosylated sample. Each lane contains 15 μ g protein. (b) Western blot analysis of TEX101 polypeptide treated with PIPLC and immunostained with anti-TEX101. Lanes 1 and 2 display the untreated plasma membranes (control) of pellet and supernatant fractions, respectively. TEX101 is present in the pellet fraction (lane 1). Lanes 3 and 4 exhibit the pellet and the supernatant fractions of PIPLC treated plasma membranes, respectively. Note that there is a complete release of TEX101 polypeptide in the supernatant fraction (lane 4) of PIPLC treated plasma membranes. The amount of plasma membrane proteins employed in the control and PIPLC treated experiments was 15 μ g.

    Journal: Biochemistry Research International

    Article Title: Identification and Characterization of TEX101 in Bovine Epididymal Spermatozoa

    doi: 10.1155/2014/573293

    Figure Lengend Snippet: (a) Immunoblots of cauda sperm plasma membrane fraction treated with N-glycanase analyzed by reducing SDS-PAGE on 15% gels and immunostained with anti-TEX101. Lane 1 represents the untreated TEX101 polypeptide, and lane 2 displays N-glycanase treated TEX101 polypeptide. Note the reduction in molecular weight (~17 kDa) of the deglycosylated sample. Each lane contains 15 μ g protein. (b) Western blot analysis of TEX101 polypeptide treated with PIPLC and immunostained with anti-TEX101. Lanes 1 and 2 display the untreated plasma membranes (control) of pellet and supernatant fractions, respectively. TEX101 is present in the pellet fraction (lane 1). Lanes 3 and 4 exhibit the pellet and the supernatant fractions of PIPLC treated plasma membranes, respectively. Note that there is a complete release of TEX101 polypeptide in the supernatant fraction (lane 4) of PIPLC treated plasma membranes. The amount of plasma membrane proteins employed in the control and PIPLC treated experiments was 15 μ g.

    Article Snippet: Enzymatic Digestion of Cauda Sperm Plasma Membrane The cauda sperm plasma membrane was treated with N-glycanase (New England Biolabs, Ipswich, MA, USA), an endoenzyme that cleaves all N-linked oligosaccharide chains from glycoproteins for 24 hours at 37°C [ ].

    Techniques: Western Blot, SDS Page, Molecular Weight

    N -Glycosylation altered the molecular mass and biochemical properties of LILRA3. A , Western blotting of PNGase F-treated ( P +) and PNGase F-untreated ( P −) rLILRA3 from 293T cells and  P. pastoris  using anti-LILRA3 mAb showed a substantial reduction in molecular mass of both eukaryotic cell-produced recombinant proteins following deglycosylation. Recombinant LILRA3 produced in  E. coli  served as a control for non-glycosylated protein. Non-PNGase F-treated LILRA3 from native macrophages ( Macs ) of two individual donors ( lanes 1  and  2 ) is shown as positive references to optimally glycosylated protein.  B , silver staining of two-dimensional gel of non-deglycosylated purified rLILRA3 from 293T cells showed a spectrum of isoelectric focusing with pI ranging from 6 to 9 ( upper panel ), but upon deglycosylation using PNGase F, it was reduced to a single focus with a pI of 7 ( lower panel ).

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M113.478578

    Figure Lengend Snippet: N -Glycosylation altered the molecular mass and biochemical properties of LILRA3. A , Western blotting of PNGase F-treated ( P +) and PNGase F-untreated ( P −) rLILRA3 from 293T cells and P. pastoris using anti-LILRA3 mAb showed a substantial reduction in molecular mass of both eukaryotic cell-produced recombinant proteins following deglycosylation. Recombinant LILRA3 produced in E. coli served as a control for non-glycosylated protein. Non-PNGase F-treated LILRA3 from native macrophages ( Macs ) of two individual donors ( lanes 1 and 2 ) is shown as positive references to optimally glycosylated protein. B , silver staining of two-dimensional gel of non-deglycosylated purified rLILRA3 from 293T cells showed a spectrum of isoelectric focusing with pI ranging from 6 to 9 ( upper panel ), but upon deglycosylation using PNGase F, it was reduced to a single focus with a pI of 7 ( lower panel ).

    Article Snippet: To determine the extent of LILRA3 glycosylation, the mammalian cell- and yeast-produced recombinant proteins were treated with PNGase F according to the manufacturer's instructions (New England Biolabs).

    Techniques: Western Blot, Produced, Recombinant, Magnetic Cell Separation, Silver Staining, Two-Dimensional Gel Electrophoresis, Purification

    Representative nano-LC-MS/MS of PNGase F-deglycosylated tryptic-digested peptides of mammalian rLILRA3 confirmed four predicted  N -glycosylation sites. A , in-gel peptide digestion of deglycosylated rLILRA3 with Glu-C showed deamidation of asparagine to aspartic acid at Asn 140  ( panel i ), Asn 281  ( panel ii ), and Asn 431  ( panel iii ) indicating  N -linked glycosylation of these sites.  B , digestion with chymotrypsin showed deamidation at Asn 281  ( panel i ) and Asn 341  ( panel ii ).  C , digestion with trypsin detected Asn 281  ( panel i ) and Asn 431  ( panel ii ). It is noteworthy that some sites were detected in peptides digested by more than one enzyme, and none of the enzymes provided full peptide coverage. The predicted Asn 302  was not detected. The sequence of the peptide, the fragmentation pattern, and the detected fragment ions are shown at the  top right  in each panel.  b  ions contain the N-terminal region of the peptide;  y  ions contain the C-terminal region of the peptide. Deamidation of asparagine to aspartic acid is designated as “ N ” with an  underscore .

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M113.478578

    Figure Lengend Snippet: Representative nano-LC-MS/MS of PNGase F-deglycosylated tryptic-digested peptides of mammalian rLILRA3 confirmed four predicted N -glycosylation sites. A , in-gel peptide digestion of deglycosylated rLILRA3 with Glu-C showed deamidation of asparagine to aspartic acid at Asn 140 ( panel i ), Asn 281 ( panel ii ), and Asn 431 ( panel iii ) indicating N -linked glycosylation of these sites. B , digestion with chymotrypsin showed deamidation at Asn 281 ( panel i ) and Asn 341 ( panel ii ). C , digestion with trypsin detected Asn 281 ( panel i ) and Asn 431 ( panel ii ). It is noteworthy that some sites were detected in peptides digested by more than one enzyme, and none of the enzymes provided full peptide coverage. The predicted Asn 302 was not detected. The sequence of the peptide, the fragmentation pattern, and the detected fragment ions are shown at the top right in each panel. b ions contain the N-terminal region of the peptide; y ions contain the C-terminal region of the peptide. Deamidation of asparagine to aspartic acid is designated as “ N ” with an underscore .

    Article Snippet: To determine the extent of LILRA3 glycosylation, the mammalian cell- and yeast-produced recombinant proteins were treated with PNGase F according to the manufacturer's instructions (New England Biolabs).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing