p0702  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Endo H
    Description:
    Endo H 50 000 units
    Catalog Number:
    p0702l
    Price:
    296
    Size:
    50 000 units
    Category:
    Glycosidases
    Buy from Supplier


    Structured Review

    New England Biolabs p0702
    Endo H
    Endo H 50 000 units
    https://www.bioz.com/result/p0702/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p0702 - by Bioz Stars, 2020-08
    99/100 stars

    Images

    Related Articles

    Transfection:

    Article Title: Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease
    Article Snippet: .. Analysis of N-glycosylation and O -fucosylation ADAMTS17EA in conditioned medium from transiently transfected HEK293F cells was deglycosylated with PNGaseF or Endoglycosidase H (New England Biolabs, Ipswich, MA) according to the manufacturer’s protocol. ..

    Purification:

    Article Title: Expression of an extremely acidic ?-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence
    Article Snippet: .. Deglycosylation of CelA4F Purified CelA4F (a ~2 μg) was treated with 20 U of Endo H for 2 h at 37°C according to the supplier's instructions (New England Biolabs, Ipswich, MA, USA) and then analyzed by SDS-PAGE. .. Enzyme assay All enzymatic assays were performed in triplicate.

    Immunoprecipitation:

    Article Title: The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion
    Article Snippet: .. Deglycosylation with Endo H and PNGase F Immunoprecipitated, bead-bound m152 isoforms were mock treated or incubated with either 750U EndoH (NEB, catalog no. P0702S, Frankfurt, Germany) or 1000U PNGase F (NEB, catalog No. P0704S, Frankfurt, Germany) for 60 min at 37 °C, followed by SDS-PAGE separation and Western blot analysis. ..

    Incubation:

    Article Title: The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion
    Article Snippet: .. Deglycosylation with Endo H and PNGase F Immunoprecipitated, bead-bound m152 isoforms were mock treated or incubated with either 750U EndoH (NEB, catalog no. P0702S, Frankfurt, Germany) or 1000U PNGase F (NEB, catalog No. P0704S, Frankfurt, Germany) for 60 min at 37 °C, followed by SDS-PAGE separation and Western blot analysis. ..

    Western Blot:

    Article Title: Rab6 Dependent Post-Golgi Trafficking of HSV1 Envelope Proteins to Sites of Virus Envelopment
    Article Snippet: .. Endo H treatment of virus glycoproteins Cells were solublized in a buffer comprising 10 mm Tris/HCl (pH 7.5), 150 mm NaCl, 0.5 mm EDTA, 0.5% NP-40, digested with Endo H (NEB) according to the manufacturers instructions and analysed by SDS–PAGE and western blotting. .. Immunofluorescence Cells for immunofluorescence were grown on coverslips and fixed with 4% paraformaldehyde in PBS for 20 min followed by permeabilization with 0.5% triton-X100 for 10 min. Endocytic structures were labelled by incubating cells with texas red conjugated transferrin (Invitrogen) at concentrations of 0.5 mg/mL in DMEM prior to fixation.

    Article Title: The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion
    Article Snippet: .. Deglycosylation with Endo H and PNGase F Immunoprecipitated, bead-bound m152 isoforms were mock treated or incubated with either 750U EndoH (NEB, catalog no. P0702S, Frankfurt, Germany) or 1000U PNGase F (NEB, catalog No. P0704S, Frankfurt, Germany) for 60 min at 37 °C, followed by SDS-PAGE separation and Western blot analysis. ..

    Article Title: CEACAM1 dampens antitumor immunity by down-regulating NKG2D ligand expression on tumor cells
    Article Snippet: .. For Rae-1 deglycosylation, the immunoprecipitates were denatured, treated with Endo-H or PNGase F according to the manufacturer’s instructions (New England Biolabs), and analyzed by Western blotting with anti–Rae-1–specific antibody (R & D Systems). .. For cell surface immunoprecipitation, cells were incubated with anti–Rae-1 on ice for 1 h and free antibodies were removed by extensive washing with PBS, lysed, and precipitated by adding protein G–conjugated Sepharose beads.

    Recombinant:

    Article Title: Complementation of a pathogenic IFNGR2 misfolding mutation with modifiers of N-glycosylation
    Article Snippet: .. We used recombinant nonglycosylated human IFN-γ (Imukin), recombinant IFN-α2b (R & D Systems), anti-V5 antibody (1:5,000; Invitrogen), Endo-H (New England Biolabs, Inc.), PNGase-F (New England Biolabs, Inc.), NB-DNJ (Sigma-Aldrich), castanospermine (Sigma-Aldrich), MG132 (Sigma-Aldrich), cycloheximide (Sigma-Aldrich), streptavidin-agarose (Invitrogen), PE-conjugated mouse anti–HLA-DR antibody (Becton Dickinson), and horseradish peroxidase (HRP)–labeled anti–mouse Ig antibody (1:10,000; GE Healthcare), NB-DNJ, and australine (Qbiogene). ..

    SDS Page:

    Article Title: Rab6 Dependent Post-Golgi Trafficking of HSV1 Envelope Proteins to Sites of Virus Envelopment
    Article Snippet: .. Endo H treatment of virus glycoproteins Cells were solublized in a buffer comprising 10 mm Tris/HCl (pH 7.5), 150 mm NaCl, 0.5 mm EDTA, 0.5% NP-40, digested with Endo H (NEB) according to the manufacturers instructions and analysed by SDS–PAGE and western blotting. .. Immunofluorescence Cells for immunofluorescence were grown on coverslips and fixed with 4% paraformaldehyde in PBS for 20 min followed by permeabilization with 0.5% triton-X100 for 10 min. Endocytic structures were labelled by incubating cells with texas red conjugated transferrin (Invitrogen) at concentrations of 0.5 mg/mL in DMEM prior to fixation.

    Article Title: The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion
    Article Snippet: .. Deglycosylation with Endo H and PNGase F Immunoprecipitated, bead-bound m152 isoforms were mock treated or incubated with either 750U EndoH (NEB, catalog no. P0702S, Frankfurt, Germany) or 1000U PNGase F (NEB, catalog No. P0704S, Frankfurt, Germany) for 60 min at 37 °C, followed by SDS-PAGE separation and Western blot analysis. ..

    Article Title: Expression of an extremely acidic ?-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence
    Article Snippet: .. Deglycosylation of CelA4F Purified CelA4F (a ~2 μg) was treated with 20 U of Endo H for 2 h at 37°C according to the supplier's instructions (New England Biolabs, Ipswich, MA, USA) and then analyzed by SDS-PAGE. .. Enzyme assay All enzymatic assays were performed in triplicate.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs endoglycosidase h
    ADAMTS17 is N -glycosylated and O -fucosylated and requires O -fucosylation for secretion. ( a ) Predicted N-glycosylation (branched stems) and O -fucosylation sites (asterisks) in ADAMTS17 and constructs used for LC-MS/MS and the ADAMTS17 secretion analysis. ( b ) Western blot analysis of ADAMTS17 EA -containing medium (Med) with or without treatment with peptide-N-glycosidase F (PNGaseF) or <t>endoglycosidase</t> H (EndoH). M = mature enzyme; Z = zymogen. For the full-length western blot see Supplemental Fig. 5a . ( c ) Schematic of TSR O -fucosylation depicting the enzymes and substrates involved. ( d ) Extracted ion chromatograms of the ions corresponding to unmodified (black), O -fucose (red), and O -fucose-glucose (blue) glycoforms of peptides from ADAMTS17 TSRs as identified by nano-LC-MS/MS. The O -fucosylation consensus sequence of the respective TSR and the modified residue (blue) are indicated above each chromatogram. ( e ) Western blot analysis of medium (Med) and lysate (Lys) from HEK293T cells with inactivated B3GLCT or POFUT2 (null) and transiently transfected with ADAMTS17-1C (1C), ADAMTS17-25P (25P) and ADAMTS17 EA (red). Co-transfected IgG (green) was used as the secretion control for quantification since it does not contain TSRs. For the full-length western blots see Supplemental Fig. 5b ,c. ( f ) Quantification of the integrated densities of the respective bands normalized to IgG (n = 3). Statistical significance was calculated using a two-sided Student t-test and compared to the band intensity measured after secretion from wild-type cells.
    Endoglycosidase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/endoglycosidase h/product/New England Biolabs
    Average 99 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    endoglycosidase h - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    ADAMTS17 is N -glycosylated and O -fucosylated and requires O -fucosylation for secretion. ( a ) Predicted N-glycosylation (branched stems) and O -fucosylation sites (asterisks) in ADAMTS17 and constructs used for LC-MS/MS and the ADAMTS17 secretion analysis. ( b ) Western blot analysis of ADAMTS17 EA -containing medium (Med) with or without treatment with peptide-N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH). M = mature enzyme; Z = zymogen. For the full-length western blot see Supplemental Fig. 5a . ( c ) Schematic of TSR O -fucosylation depicting the enzymes and substrates involved. ( d ) Extracted ion chromatograms of the ions corresponding to unmodified (black), O -fucose (red), and O -fucose-glucose (blue) glycoforms of peptides from ADAMTS17 TSRs as identified by nano-LC-MS/MS. The O -fucosylation consensus sequence of the respective TSR and the modified residue (blue) are indicated above each chromatogram. ( e ) Western blot analysis of medium (Med) and lysate (Lys) from HEK293T cells with inactivated B3GLCT or POFUT2 (null) and transiently transfected with ADAMTS17-1C (1C), ADAMTS17-25P (25P) and ADAMTS17 EA (red). Co-transfected IgG (green) was used as the secretion control for quantification since it does not contain TSRs. For the full-length western blots see Supplemental Fig. 5b ,c. ( f ) Quantification of the integrated densities of the respective bands normalized to IgG (n = 3). Statistical significance was calculated using a two-sided Student t-test and compared to the band intensity measured after secretion from wild-type cells.

    Journal: Scientific Reports

    Article Title: Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease

    doi: 10.1038/srep41871

    Figure Lengend Snippet: ADAMTS17 is N -glycosylated and O -fucosylated and requires O -fucosylation for secretion. ( a ) Predicted N-glycosylation (branched stems) and O -fucosylation sites (asterisks) in ADAMTS17 and constructs used for LC-MS/MS and the ADAMTS17 secretion analysis. ( b ) Western blot analysis of ADAMTS17 EA -containing medium (Med) with or without treatment with peptide-N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH). M = mature enzyme; Z = zymogen. For the full-length western blot see Supplemental Fig. 5a . ( c ) Schematic of TSR O -fucosylation depicting the enzymes and substrates involved. ( d ) Extracted ion chromatograms of the ions corresponding to unmodified (black), O -fucose (red), and O -fucose-glucose (blue) glycoforms of peptides from ADAMTS17 TSRs as identified by nano-LC-MS/MS. The O -fucosylation consensus sequence of the respective TSR and the modified residue (blue) are indicated above each chromatogram. ( e ) Western blot analysis of medium (Med) and lysate (Lys) from HEK293T cells with inactivated B3GLCT or POFUT2 (null) and transiently transfected with ADAMTS17-1C (1C), ADAMTS17-25P (25P) and ADAMTS17 EA (red). Co-transfected IgG (green) was used as the secretion control for quantification since it does not contain TSRs. For the full-length western blots see Supplemental Fig. 5b ,c. ( f ) Quantification of the integrated densities of the respective bands normalized to IgG (n = 3). Statistical significance was calculated using a two-sided Student t-test and compared to the band intensity measured after secretion from wild-type cells.

    Article Snippet: Analysis of N-glycosylation and O -fucosylation ADAMTS17EA in conditioned medium from transiently transfected HEK293F cells was deglycosylated with PNGaseF or Endoglycosidase H (New England Biolabs, Ipswich, MA) according to the manufacturer’s protocol.

    Techniques: Construct, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Sequencing, Modification, Transfection

    Rab6 depletion inhibits virus production at late stages of infection. A) HeLa cells transfected with neg, Rab1 or Rab6 siRNAs were infected with HSV1 2 days later and harvested 16 h after infection for western blotting with antibodies as indicated. Mature glycosylated forms of gD and gE are marked by asterisk. B) The status of glycoprotein glycosylation in control (neg), Rab6 depleted or monensin treated cells was determined by Endo H treatment of infected cell lysates harvested at 16 h, and analysed by western blotting for gD. Three isoforms of gD are denoted by 1, 2 3 . C) The level of DNA replication in control (neg), Rab6 depleted or Ara C treated cells was analysed by semi-quantitative PCR using a primer pair specific for the UL47 gene. PCR cycle numbers are denoted. D) Hela cells transfected with neg or Rab6 siRNAs were infected 2 days later with HSV1, and cell-associated and released virus titrated on Vero cells. Molecular weight markers are shown in kDa.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Rab6 Dependent Post-Golgi Trafficking of HSV1 Envelope Proteins to Sites of Virus Envelopment

    doi: 10.1111/tra.12134

    Figure Lengend Snippet: Rab6 depletion inhibits virus production at late stages of infection. A) HeLa cells transfected with neg, Rab1 or Rab6 siRNAs were infected with HSV1 2 days later and harvested 16 h after infection for western blotting with antibodies as indicated. Mature glycosylated forms of gD and gE are marked by asterisk. B) The status of glycoprotein glycosylation in control (neg), Rab6 depleted or monensin treated cells was determined by Endo H treatment of infected cell lysates harvested at 16 h, and analysed by western blotting for gD. Three isoforms of gD are denoted by 1, 2 3 . C) The level of DNA replication in control (neg), Rab6 depleted or Ara C treated cells was analysed by semi-quantitative PCR using a primer pair specific for the UL47 gene. PCR cycle numbers are denoted. D) Hela cells transfected with neg or Rab6 siRNAs were infected 2 days later with HSV1, and cell-associated and released virus titrated on Vero cells. Molecular weight markers are shown in kDa.

    Article Snippet: Endo H treatment of virus glycoproteins Cells were solublized in a buffer comprising 10 mm Tris/HCl (pH 7.5), 150 mm NaCl, 0.5 mm EDTA, 0.5% NP-40, digested with Endo H (NEB) according to the manufacturers instructions and analysed by SDS–PAGE and western blotting.

    Techniques: Infection, Transfection, Western Blot, Acetylene Reduction Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Molecular Weight

    A single polar uncharged residue in IBV E is required for disruption of cargo trafficking. (A) A helical wheel diagram of the HD of IBV E. Polar uncharged residues are shown in blue; residues mutated to alanine are outlined in red. (B) An immunoblot shows that the alanine mutants of IBV E are expressed and run at a similar molecular weight when transiently expressed in HeLa cells. (C) VSV G was transiently co-expressed with the indicated protein in HeLa cells. 18–22 hours after transfection the cells were pulse-labeled with 35 S-methionine/cysteine and chased for 0, 25, and 50 min. VSV G was immunoprecipitated from each sample and digested with endoglycosidase H. The mature (**) and immature (*) forms are indicated. Data from control, IBV E, S13A, and T16 A is shown. (D) Quantification of (C) showing that the T16A mutation inactivates the trafficking block. At each time-point the signal intensity for the mature and immature bands was measured. The percent of endo H resistant VSV G was calculated by dividing the signal for the mature band by the total signal (mature+immature). Data are from at least two independent experiments. Error bars represent +/− SEM.

    Journal: PLoS Pathogens

    Article Title: A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

    doi: 10.1371/journal.ppat.1002674

    Figure Lengend Snippet: A single polar uncharged residue in IBV E is required for disruption of cargo trafficking. (A) A helical wheel diagram of the HD of IBV E. Polar uncharged residues are shown in blue; residues mutated to alanine are outlined in red. (B) An immunoblot shows that the alanine mutants of IBV E are expressed and run at a similar molecular weight when transiently expressed in HeLa cells. (C) VSV G was transiently co-expressed with the indicated protein in HeLa cells. 18–22 hours after transfection the cells were pulse-labeled with 35 S-methionine/cysteine and chased for 0, 25, and 50 min. VSV G was immunoprecipitated from each sample and digested with endoglycosidase H. The mature (**) and immature (*) forms are indicated. Data from control, IBV E, S13A, and T16 A is shown. (D) Quantification of (C) showing that the T16A mutation inactivates the trafficking block. At each time-point the signal intensity for the mature and immature bands was measured. The percent of endo H resistant VSV G was calculated by dividing the signal for the mature band by the total signal (mature+immature). Data are from at least two independent experiments. Error bars represent +/− SEM.

    Article Snippet: Immune complexes were eluted in 1% SDS [pH 6.8] at 100°C and digested in 75 mM Na-citrate [pH 5.5] with 0.2 µl endo H (100 units) (New England Biolabs) at 37°C overnight.

    Techniques: Molecular Weight, Transfection, Labeling, Immunoprecipitation, Mutagenesis, Blocking Assay

    The transmembrane topology of IBV E promotes disruption of protein trafficking. (A) VSV G pulse-chase coupled with endo H digestion as described in Figure 1 . The mature (**) and immature (*) forms are indicated. (B) Quantification of the pulse-chase data. Both ssIBV E and IBV E dramatically affect protein trafficking, while FLAG-IBV E has a more modest effect. Data are from 3 independent experiments. Error bars represent +/− SEM.

    Journal: PLoS Pathogens

    Article Title: A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

    doi: 10.1371/journal.ppat.1002674

    Figure Lengend Snippet: The transmembrane topology of IBV E promotes disruption of protein trafficking. (A) VSV G pulse-chase coupled with endo H digestion as described in Figure 1 . The mature (**) and immature (*) forms are indicated. (B) Quantification of the pulse-chase data. Both ssIBV E and IBV E dramatically affect protein trafficking, while FLAG-IBV E has a more modest effect. Data are from 3 independent experiments. Error bars represent +/− SEM.

    Article Snippet: Immune complexes were eluted in 1% SDS [pH 6.8] at 100°C and digested in 75 mM Na-citrate [pH 5.5] with 0.2 µl endo H (100 units) (New England Biolabs) at 37°C overnight.

    Techniques: Pulse Chase

    Substitution with other polar uncharged residues is not tolerated at position 16. (A) Multiple sequence alignment of CoV E proteins. Negatively charged residues are colored red, positively charged residues are colored in blue and polar uncharged residues are colored in yellow. The box encompasses the hydrophobic domain of IBV E, and the arrow denotes position 16 in IBV E. (B) VSV G pulse-chase coupled with endo H digestion as described in Figure 1 . Mutation of T16 to S, N, or Q does not restore the ability of the protein to disrupt trafficking of VSV G through the Golgi complex. Data are from at least two independent experiments. Error bars represent +/− SEM. (C) IBV E protein with S, N or Q substituted for T16 does not induce Golgi complex disassembly (See Figure 2 for description of quantification). Data are from 3 independent experiments, N≥48 for each condition. Error bars represent +/− SEM, and the asterisk denotes a significant increase in Golgi disruption compared to the control by Student's t -test (p≤3×10 −5 ).

    Journal: PLoS Pathogens

    Article Title: A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

    doi: 10.1371/journal.ppat.1002674

    Figure Lengend Snippet: Substitution with other polar uncharged residues is not tolerated at position 16. (A) Multiple sequence alignment of CoV E proteins. Negatively charged residues are colored red, positively charged residues are colored in blue and polar uncharged residues are colored in yellow. The box encompasses the hydrophobic domain of IBV E, and the arrow denotes position 16 in IBV E. (B) VSV G pulse-chase coupled with endo H digestion as described in Figure 1 . Mutation of T16 to S, N, or Q does not restore the ability of the protein to disrupt trafficking of VSV G through the Golgi complex. Data are from at least two independent experiments. Error bars represent +/− SEM. (C) IBV E protein with S, N or Q substituted for T16 does not induce Golgi complex disassembly (See Figure 2 for description of quantification). Data are from 3 independent experiments, N≥48 for each condition. Error bars represent +/− SEM, and the asterisk denotes a significant increase in Golgi disruption compared to the control by Student's t -test (p≤3×10 −5 ).

    Article Snippet: Immune complexes were eluted in 1% SDS [pH 6.8] at 100°C and digested in 75 mM Na-citrate [pH 5.5] with 0.2 µl endo H (100 units) (New England Biolabs) at 37°C overnight.

    Techniques: Sequencing, Pulse Chase, Mutagenesis

    CEACAM1 mediates intracellular retention of NKG2DL. (a) Flow cytometric analysis shows cell surface and intracellular Rae-1 expression in indicated tumor cells. (b) Fluorescence microscopy analysis shows Rae-1(red) localization detected by goat anti-Rae1 followed by rhodamine-conjugated mouse anti–goat IgG in CEACAM1-silenced (CC1shRNA) and nonsilenced (nonTshRNA) MC38 tumor cells (bars: left two panels, 100 mm; right panel, 20 mm). (c) Immunoblot (IB) shows Rae-1 structure in indicated tumor cells (H, high molecular weight; L, low molecular weight). (d) Immunoblot analysis shows MHC class I expression in CEACAM1-silenced (CC1shRNA) and nonsilenced (nonTshRNA) MC38 tumor cells. (e) CEACAM1-silenced (CC1shRNA) and nonsilenced (nonTshRNA) cells were labeled with membrane impermeable biotin. Rae-1 was precipitated with Rae-1–specific antibody (T-IP) or streptavidin (B-IP) and detected with Rae-1–specific antibody. (f) The immunoblot (IB) shows the patterns of Rae-1 immunoprecipitated (IP) from CEACAM1-silenced and nonsilenced cell lysates followed by mock treatment (CTL: control) or treatment with either Endo-H or PNGase F (PNG-F). All results are representative of three or four independent experiments, respectively.

    Journal: The Journal of Experimental Medicine

    Article Title: CEACAM1 dampens antitumor immunity by down-regulating NKG2D ligand expression on tumor cells

    doi: 10.1084/jem.20102575

    Figure Lengend Snippet: CEACAM1 mediates intracellular retention of NKG2DL. (a) Flow cytometric analysis shows cell surface and intracellular Rae-1 expression in indicated tumor cells. (b) Fluorescence microscopy analysis shows Rae-1(red) localization detected by goat anti-Rae1 followed by rhodamine-conjugated mouse anti–goat IgG in CEACAM1-silenced (CC1shRNA) and nonsilenced (nonTshRNA) MC38 tumor cells (bars: left two panels, 100 mm; right panel, 20 mm). (c) Immunoblot (IB) shows Rae-1 structure in indicated tumor cells (H, high molecular weight; L, low molecular weight). (d) Immunoblot analysis shows MHC class I expression in CEACAM1-silenced (CC1shRNA) and nonsilenced (nonTshRNA) MC38 tumor cells. (e) CEACAM1-silenced (CC1shRNA) and nonsilenced (nonTshRNA) cells were labeled with membrane impermeable biotin. Rae-1 was precipitated with Rae-1–specific antibody (T-IP) or streptavidin (B-IP) and detected with Rae-1–specific antibody. (f) The immunoblot (IB) shows the patterns of Rae-1 immunoprecipitated (IP) from CEACAM1-silenced and nonsilenced cell lysates followed by mock treatment (CTL: control) or treatment with either Endo-H or PNGase F (PNG-F). All results are representative of three or four independent experiments, respectively.

    Article Snippet: For Rae-1 deglycosylation, the immunoprecipitates were denatured, treated with Endo-H or PNGase F according to the manufacturer’s instructions (New England Biolabs), and analyzed by Western blotting with anti–Rae-1–specific antibody (R & D Systems).

    Techniques: Flow Cytometry, Expressing, Fluorescence, Microscopy, Molecular Weight, Labeling, Immunoprecipitation, CTL Assay