endoglycosidaseh  (New England Biolabs)


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    O Glycosidase
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    O Glycosidase 10 000 000 units
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    New England Biolabs endoglycosidaseh
    O Glycosidase
    O Glycosidase 10 000 000 units
    https://www.bioz.com/result/endoglycosidaseh/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
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    endoglycosidaseh - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis"

    Article Title: Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.198655

    The large N-terminal domain of the type I TMP VCAM1 results in a complete block of its membrane integration by mycolactone. (A) VCAM1 translated in the absence or presence of mycolactone (MYC) and treated with EndoglycosidaseH (EndoH). (B) VCAM1 and VCAM1 60 constructs (wild type and S707L/S707L* mutants) used in this study. (C) VCAM1 60 and a version containing an artificial N-glycosylation site (C52N) translated in the absence or presence of mycolactone, without or without EndoH. (D) VCAM1 60 and a variant with a more hydrophobic TMD (VCAM1 60 S707L* ) translated in the absence or presence of mycolactone, without or without subsequent EndoH treatment. Estimated TMD hydrophobicities (kcal/mol) are indicated in D. Graph shows the reduction in the amount of ‘+g’ VCAM1 60 and VCAM1 60 S707L* in the presence of mycolactone, relative to control samples, as described in the legend to Fig. 1 . is also shown in D (graph). The statistical test performed was one-way ANOVA. Error bars show mean±s.d. VCAM1, n =3 VCAM1 60 , n =4; VCAM1 60 S707L* , n =3). P -values are as defined in Fig. 1 legend. (E) Translation of VCAM1, VCAM1 60 and the secretory protein cecropin, possessing a C-terminal opsin tag (CecOPG2), performed with increasing concentrations of CAM741 or an equivalent volume of DMSO (‘−’). (F) VCAM1 60 and VCAM1 60 S707L* translated in the absence or presence of 250 nM CAM741. Other symbols are as defined in Fig. 1 legend.
    Figure Legend Snippet: The large N-terminal domain of the type I TMP VCAM1 results in a complete block of its membrane integration by mycolactone. (A) VCAM1 translated in the absence or presence of mycolactone (MYC) and treated with EndoglycosidaseH (EndoH). (B) VCAM1 and VCAM1 60 constructs (wild type and S707L/S707L* mutants) used in this study. (C) VCAM1 60 and a version containing an artificial N-glycosylation site (C52N) translated in the absence or presence of mycolactone, without or without EndoH. (D) VCAM1 60 and a variant with a more hydrophobic TMD (VCAM1 60 S707L* ) translated in the absence or presence of mycolactone, without or without subsequent EndoH treatment. Estimated TMD hydrophobicities (kcal/mol) are indicated in D. Graph shows the reduction in the amount of ‘+g’ VCAM1 60 and VCAM1 60 S707L* in the presence of mycolactone, relative to control samples, as described in the legend to Fig. 1 . is also shown in D (graph). The statistical test performed was one-way ANOVA. Error bars show mean±s.d. VCAM1, n =3 VCAM1 60 , n =4; VCAM1 60 S707L* , n =3). P -values are as defined in Fig. 1 legend. (E) Translation of VCAM1, VCAM1 60 and the secretory protein cecropin, possessing a C-terminal opsin tag (CecOPG2), performed with increasing concentrations of CAM741 or an equivalent volume of DMSO (‘−’). (F) VCAM1 60 and VCAM1 60 S707L* translated in the absence or presence of 250 nM CAM741. Other symbols are as defined in Fig. 1 legend.

    Techniques Used: Blocking Assay, Construct, Variant Assay

    ER integration of the type I TMP CD3δ in the presence of mycolactone is driven by its TMD. (A) Truncated mRNAs coding for CD3δ (top panel) and CD3δ D111L (bottom panel) and lacking stop codons translated in the absence or presence of mycolactone (MYC) without puromycin-mediated release. The nascent chain length of each truncation is shown, as well as the number of residues synthesised C-terminal to the TMD to provide an estimate of its distance from the peptidyl-transferase centre (PTC) of the ribosome. Truncations where all or part of the TMD is likely obscured by the ribosomal exit tunnel (based on Cabrita et al., 2016 ) are indicated by the bracketed area. CD3δ 158 is encompassed by a dashed bracket, since its TMD is likely on the border of having just fully emerged from the ribosomal exit tunnel. Arrowheads indicate maximal glycosylation resulting from the TMD-dependent rescue of integration in the presence of mycolactone. (B) Versions of (i) CD3δ and (ii) CD3δ D111L lacking signal sequences (ΔSS) translated in the absence and presence of mycolactone, without or without subsequent EndoglycosidaseH (EndoH) treatment. (C) Predicted mechanism of type I TMP integration in the absence (i) and presence (ii) of mycolactone. Other symbols are as defined in Fig. 1 legend. ‘+g’, glycosylated; ‘0g’, non-glycosylated; ‘C’, C-terminus; FL, full length; ‘N’, N-terminus.
    Figure Legend Snippet: ER integration of the type I TMP CD3δ in the presence of mycolactone is driven by its TMD. (A) Truncated mRNAs coding for CD3δ (top panel) and CD3δ D111L (bottom panel) and lacking stop codons translated in the absence or presence of mycolactone (MYC) without puromycin-mediated release. The nascent chain length of each truncation is shown, as well as the number of residues synthesised C-terminal to the TMD to provide an estimate of its distance from the peptidyl-transferase centre (PTC) of the ribosome. Truncations where all or part of the TMD is likely obscured by the ribosomal exit tunnel (based on Cabrita et al., 2016 ) are indicated by the bracketed area. CD3δ 158 is encompassed by a dashed bracket, since its TMD is likely on the border of having just fully emerged from the ribosomal exit tunnel. Arrowheads indicate maximal glycosylation resulting from the TMD-dependent rescue of integration in the presence of mycolactone. (B) Versions of (i) CD3δ and (ii) CD3δ D111L lacking signal sequences (ΔSS) translated in the absence and presence of mycolactone, without or without subsequent EndoglycosidaseH (EndoH) treatment. (C) Predicted mechanism of type I TMP integration in the absence (i) and presence (ii) of mycolactone. Other symbols are as defined in Fig. 1 legend. ‘+g’, glycosylated; ‘0g’, non-glycosylated; ‘C’, C-terminus; FL, full length; ‘N’, N-terminus.

    Techniques Used:

    Mycolactone does not interfere with type III TMP integration. (A) Translation of GypC in the absence or presence of mycolactone (MYC), followed by subsequent treatment with EndoglycosidaseH (EndoH). (B) Graph shows change in the amount of glycosylated (+g) GypC and related constructs in the presence of mycolactone, relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. GypC, n =10; others, n =3. Ns, not significant. (C) Estimated TMD hydrophobicities (kcal/mol) of GypC and related constructs. (D) Translation of two variants of GypC with reduced TMD hydrophobicity. (E) GypC truncations lacking stop codons. For crosslinking experiments, truncations contained a single artificially introduced cysteine residue at either position 52 or 84, as denoted by an asterisk. (F) Truncated GypC chains synthesised in the absence or presence of mycolactone without puromycin-mediated release. The glycosylation of nascent chains when still attached to the ribosome (indicated by ‘peptRNA’) was observed. (G) Truncated GypC chains containing a single cysteine residue [either *(52) or *(84)] synthesised in the absence or presence of mycolactone without puromycin-mediated release to generate membrane integration intermediates. Samples were treated with the crosslinking reagent BMH, subjected to extraction with alkaline sodium carbonate, and analysed by SDS-PAGE. Adducts between the nascent chain and Sec61β (xSec61β) or the nascent chain and Sec61α/Sec61α and Sec61β (xSec61α/αβ) are indicated (see also Fig. S3B ). Mycolactone-sensitive adducts are indicated by arrowheads. Other symbols are as defined in Fig. 1 legend. FL, full length.
    Figure Legend Snippet: Mycolactone does not interfere with type III TMP integration. (A) Translation of GypC in the absence or presence of mycolactone (MYC), followed by subsequent treatment with EndoglycosidaseH (EndoH). (B) Graph shows change in the amount of glycosylated (+g) GypC and related constructs in the presence of mycolactone, relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. GypC, n =10; others, n =3. Ns, not significant. (C) Estimated TMD hydrophobicities (kcal/mol) of GypC and related constructs. (D) Translation of two variants of GypC with reduced TMD hydrophobicity. (E) GypC truncations lacking stop codons. For crosslinking experiments, truncations contained a single artificially introduced cysteine residue at either position 52 or 84, as denoted by an asterisk. (F) Truncated GypC chains synthesised in the absence or presence of mycolactone without puromycin-mediated release. The glycosylation of nascent chains when still attached to the ribosome (indicated by ‘peptRNA’) was observed. (G) Truncated GypC chains containing a single cysteine residue [either *(52) or *(84)] synthesised in the absence or presence of mycolactone without puromycin-mediated release to generate membrane integration intermediates. Samples were treated with the crosslinking reagent BMH, subjected to extraction with alkaline sodium carbonate, and analysed by SDS-PAGE. Adducts between the nascent chain and Sec61β (xSec61β) or the nascent chain and Sec61α/Sec61α and Sec61β (xSec61α/αβ) are indicated (see also Fig. S3B ). Mycolactone-sensitive adducts are indicated by arrowheads. Other symbols are as defined in Fig. 1 legend. FL, full length.

    Techniques Used: Construct, SDS Page

    Mycolactone traps headfirst-inserting type II TMPs in an N-lumenal–C-cytosolic topology. (A) ASGPR H1 and ASGPR H1Δ. Translation of ASGPR H1 (B) and ASGPR H1Δ (C) performed in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Membrane fractions were subjected to extraction with alkaline sodium carbonate prior to analysis. (D) Graph shows the amount of glycosylated (‘+g’) and non-glycosylated (‘0g’) ASGPR H1 and ASGPR H1Δ in the presence of mycolactone, relative to control samples. These values were determined by dividing the quantity of ‘+g’ or ‘0g’ substrate obtained in the presence of mycolactone by the quantity of '+g' or '0g' substrate obtained in the absence of mycolactone and are expressed as percentages. Dashed red line represents the value for comparative material for samples treated with a vehicle control. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. (E) Diagram showing type II TMPs that insert using a hairpin mechanism (i) or a headfirst/inversion mechanism (ii), as well as the headfirst insertion of type III TMPs (iii). Faded steps represent those that are prevented by mycolactone. Dashed arrow shows the predicted route taken by headfirst-inserting type II TMPs when inversion is prevented by mycolactone.
    Figure Legend Snippet: Mycolactone traps headfirst-inserting type II TMPs in an N-lumenal–C-cytosolic topology. (A) ASGPR H1 and ASGPR H1Δ. Translation of ASGPR H1 (B) and ASGPR H1Δ (C) performed in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Membrane fractions were subjected to extraction with alkaline sodium carbonate prior to analysis. (D) Graph shows the amount of glycosylated (‘+g’) and non-glycosylated (‘0g’) ASGPR H1 and ASGPR H1Δ in the presence of mycolactone, relative to control samples. These values were determined by dividing the quantity of ‘+g’ or ‘0g’ substrate obtained in the presence of mycolactone by the quantity of '+g' or '0g' substrate obtained in the absence of mycolactone and are expressed as percentages. Dashed red line represents the value for comparative material for samples treated with a vehicle control. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. (E) Diagram showing type II TMPs that insert using a hairpin mechanism (i) or a headfirst/inversion mechanism (ii), as well as the headfirst insertion of type III TMPs (iii). Faded steps represent those that are prevented by mycolactone. Dashed arrow shows the predicted route taken by headfirst-inserting type II TMPs when inversion is prevented by mycolactone.

    Techniques Used:

    Mycolactone sensitivity is dependent upon which TMD-flanking region is translocated. (A) A chimeric protein containing Ii downstream of a pre-prolactin (PPL) signal sequence (i) and the two topologies it might assume following integration into RMs, depending on whether the region that is translocated is N-terminal (ii) or C-terminal (iii) of the TMD. (B) Translation of PPL-Ii and PPL-Ii G47L Q48L* in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Samples were analysed following immunoprecipitation of Ii. (C) Graph showing the amount of signal-cleaved (‘sc’) or glycosylated (‘+g’) substrate in the presence of mycolactone relative to control samples. These values were determined by dividing the quantity of ‘sc’ or ‘+g’ substrate obtained in the presence of mycolactone by the quantity of ‘sc’ or ‘+g’ substrate obtained in the absence of mycolactone and are expressed as percentages. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values and other symbols are as defined in Fig. 1 legend.
    Figure Legend Snippet: Mycolactone sensitivity is dependent upon which TMD-flanking region is translocated. (A) A chimeric protein containing Ii downstream of a pre-prolactin (PPL) signal sequence (i) and the two topologies it might assume following integration into RMs, depending on whether the region that is translocated is N-terminal (ii) or C-terminal (iii) of the TMD. (B) Translation of PPL-Ii and PPL-Ii G47L Q48L* in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Samples were analysed following immunoprecipitation of Ii. (C) Graph showing the amount of signal-cleaved (‘sc’) or glycosylated (‘+g’) substrate in the presence of mycolactone relative to control samples. These values were determined by dividing the quantity of ‘sc’ or ‘+g’ substrate obtained in the presence of mycolactone by the quantity of ‘sc’ or ‘+g’ substrate obtained in the absence of mycolactone and are expressed as percentages. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values and other symbols are as defined in Fig. 1 legend.

    Techniques Used: Sequencing, Immunoprecipitation

    Mycolactone efficiently blocks type II TMP integration. (A) Full-length Ii (wild type and G47L Q48L mutant) and the Ii 125 truncation used in this study. (B) Estimated TMD hydrophobicities (kcal/mol) of Ii and Ii G47L Q48L . (C) Graph shows the reduction in the amount of glycosylated (+g) Ii and related constructs in the presence of mycolactone (MYC), relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. Translation in the absence or presence of mycolactone performed using Ii (D), Ii G47L Q48L (E) and Ii 125 (F), which was followed by treatment with EndoglycosidaseH (EndoH). (G) Ii truncations used in this study. For crosslinking experiments, truncations contained either a native cysteine residue (C28) or one that was artificially introduced [*(50)]. A truncated version of TNFα used for crosslinking analysis (as described in MacKinnon et al., 2014 ) is shown for comparative purposes. Crosslinking was performed on Ii truncations (H) and Ii 125 *(50) (I) and the resulting adducts are labelled as described in the Fig. 4 G legend. Other symbols are as defined in Fig. 1 legend. Puro, puromycin.
    Figure Legend Snippet: Mycolactone efficiently blocks type II TMP integration. (A) Full-length Ii (wild type and G47L Q48L mutant) and the Ii 125 truncation used in this study. (B) Estimated TMD hydrophobicities (kcal/mol) of Ii and Ii G47L Q48L . (C) Graph shows the reduction in the amount of glycosylated (+g) Ii and related constructs in the presence of mycolactone (MYC), relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. Translation in the absence or presence of mycolactone performed using Ii (D), Ii G47L Q48L (E) and Ii 125 (F), which was followed by treatment with EndoglycosidaseH (EndoH). (G) Ii truncations used in this study. For crosslinking experiments, truncations contained either a native cysteine residue (C28) or one that was artificially introduced [*(50)]. A truncated version of TNFα used for crosslinking analysis (as described in MacKinnon et al., 2014 ) is shown for comparative purposes. Crosslinking was performed on Ii truncations (H) and Ii 125 *(50) (I) and the resulting adducts are labelled as described in the Fig. 4 G legend. Other symbols are as defined in Fig. 1 legend. Puro, puromycin.

    Techniques Used: Mutagenesis, Construct

    2) Product Images from "Endo-?-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins *"

    Article Title: Endo-?-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M112.018119

    Endoglycosidase activity found in Bifidobacterium isolates. A , deglycosylation of RNaseB by B. infantis ATCC 15697 over time. B–D , overnight incubation with RNaseB was evaluated for other isolates of B. longum ( B ), B. infantis ( C ), or B. breve ( D ). E , phylogenetic representation of predicted endoglycosidase protein sequences found in bifidobacterial isolates. C: control reactions.
    Figure Legend Snippet: Endoglycosidase activity found in Bifidobacterium isolates. A , deglycosylation of RNaseB by B. infantis ATCC 15697 over time. B–D , overnight incubation with RNaseB was evaluated for other isolates of B. longum ( B ), B. infantis ( C ), or B. breve ( D ). E , phylogenetic representation of predicted endoglycosidase protein sequences found in bifidobacterial isolates. C: control reactions.

    Techniques Used: Activity Assay, Incubation

    3) 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 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

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    Article Snippet: For detecting HA-Rim101, Myc-Rim101-531, and genomic-encoded Rsb1-HA proteins, we enhanced the signal of the anti-HA antibody Y-11 and the anti-Myc antibody PL-14 using Can Get Signal Immunoreaction Enhancer Solution (Toyobo), according to the manufacturer's manual. .. Proteins (5 μl) in 1× SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and a trace amount of bromophenol blue containing 5% 2-mercaptoethanol) were diluted with 40 μl 0.05 M sodium citrate (pH 5.5) containing 1 mM phenylmethylsulfonyl fluoride, and treated at 37°C for 1 h with 1000 U of recombinant endoglycosidase H (Endo H; New England Biolabs, Beverly, MA). .. The samples were then mixed with 12 μl 4× SDS sample buffer and 3 μl 2-mercaptoethanol and incubated at 37°C for 5 min. A portion of each sample was separated by SDS-PAGE and subjected to immunoblotting.

    other:

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    Article Snippet: Quantification of the data was performed after digestion by endoglycosidase H (Endo H), an enzyme that specifically cleaves N-linked carbohydrates.

    Incubation:

    Article Title: A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity
    Article Snippet: Viral proteins were precipitated as described earlier ( , ), using rabbit antisera directed against recombinant FV proteins and specific for Env ( ) and Gag ( ). .. For glycosidase treatment, protein A-Sepharose eluates were denatured by boiling in 0.5% SDS–1% β-mercaptoethanol and subsequently incubated with endoglycosidase H (endo H) peptide N -glycosidase or (PNGase F) in the appropriate incubation buffer as suggested by the manufacturer (New England Biolabs) prior to loading on gels for SDS-polyacrylamide gel electrophoresis (PAGE). .. Particle-associated proteins were analyzed after centrifugation through a 20% sucrose cushion as described previously ( , ).

    Nucleic Acid Electrophoresis:

    Article Title: A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity
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    Polyacrylamide Gel Electrophoresis:

    Article Title: A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity
    Article Snippet: Viral proteins were precipitated as described earlier ( , ), using rabbit antisera directed against recombinant FV proteins and specific for Env ( ) and Gag ( ). .. For glycosidase treatment, protein A-Sepharose eluates were denatured by boiling in 0.5% SDS–1% β-mercaptoethanol and subsequently incubated with endoglycosidase H (endo H) peptide N -glycosidase or (PNGase F) in the appropriate incubation buffer as suggested by the manufacturer (New England Biolabs) prior to loading on gels for SDS-polyacrylamide gel electrophoresis (PAGE). .. Particle-associated proteins were analyzed after centrifugation through a 20% sucrose cushion as described previously ( , ).

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    New England Biolabs endoglycosidase h endo h digestion
    ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without <t>endoglycosidase</t> H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. <t>Endo</t> H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p
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    ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Journal: PLoS ONE

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    doi: 10.1371/journal.pone.0207948

    Figure Lengend Snippet: ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Article Snippet: Endoglycosidase H (endo H) digestion and Peptide-N-Glycosidase F (PNGase F) (New England Biolabs) digestion were performed according to the published procedure [ ].

    Techniques: Activation Assay, Expressing, Transfection, Incubation, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Journal: PLoS ONE

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    doi: 10.1371/journal.pone.0207948

    Figure Lengend Snippet: BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Article Snippet: Endoglycosidase H (endo H) digestion and Peptide-N-Glycosidase F (PNGase F) (New England Biolabs) digestion were performed according to the published procedure [ ].

    Techniques: Stable Transfection, Expressing, Cell Culture, SDS Page, Western Blot, Positive Control, Fluorescence, Incubation, Quantitation Assay

    Identification of FV Env subdomains by different antisera. Cell or virus lysates of 293T cells transfected with the Gag/Pol-expressing FV vector pMH62 (Gag/Pol), wild-type FV Env expression construct pcHFE EM02 (Env), or empty expression vector (pCDNA3.1) as indicated were analyzed by RIPA or Western blotting. The identity of each FV protein is indicated. The gp48 TM  protein often comigrates with an immunoreactive cellular protein present in negative controls. (A) Schematic outline of the transfected expression constructs. SFFV, spleen focus-forming virus; bgH pA, bovine growth hormone polyadenylation site. (B) RIPA of cells labeled for 20 h using polyclonal rabbit sera specific for Gag (α-Gag), SP Env /SU (α-SP/SU), or SP Env  (α-SP). A longer exposure of the lower part of the SDS–10% polyacrylamide gel with the cellular lysates is shown separately, and an autoradiogram of metabolically labeled purified FV particles separated by SDS-PAGE is shown to the right. (C) Western blot analysis (SDS-PAGE [12% gel]) of cellular lysates and purified FV particles using polyclonal rabbit sera specific for Gag (α-Gag) or SP Env  (α-SP). (D) Western blot analysis (SDS-PAGE [12% gel]) of equilibrium sedimentation gradient fractions using polyclonal rabbit sera specific for Gag (α-Gag) and SP Env  (α-SP). (E) Pulse-chase analysis of FV Env maturation. Transfected 293T cells were pulse-labeled for 30 min and then chased for different time periods as indicated at the top with fresh growth medium containing an excess of cold methionine and cysteine. Equal cell lysate samples were immunoprecipitated with FV-specific antisera as indicated to the left. Equivalent aliquots of the protein A eluates were incubated with glycosidases as indicated at the top prior to separation by SDS-PAGE (7.5% gel). Identities of the different forms (g [fully glycosylated], h [endo H resistant] and p [N-deglycosylated]) of gp130 Env  (solid arrows), gp80 SU  (shaded arrows), and gp48 TM  (open arrows) are indicated. The bands marked with asterisks at the 1- and 3-h time point after immunoprecipitation with anti-SP/SU antiserum and PNGase F treatment represent the fully N-deglycosylated form of SU running only slightly faster than the fully glycosylated form of TM.

    Journal: Journal of Virology

    Article Title: A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

    doi: 10.1128/JVI.75.13.5762-5771.2001

    Figure Lengend Snippet: Identification of FV Env subdomains by different antisera. Cell or virus lysates of 293T cells transfected with the Gag/Pol-expressing FV vector pMH62 (Gag/Pol), wild-type FV Env expression construct pcHFE EM02 (Env), or empty expression vector (pCDNA3.1) as indicated were analyzed by RIPA or Western blotting. The identity of each FV protein is indicated. The gp48 TM protein often comigrates with an immunoreactive cellular protein present in negative controls. (A) Schematic outline of the transfected expression constructs. SFFV, spleen focus-forming virus; bgH pA, bovine growth hormone polyadenylation site. (B) RIPA of cells labeled for 20 h using polyclonal rabbit sera specific for Gag (α-Gag), SP Env /SU (α-SP/SU), or SP Env (α-SP). A longer exposure of the lower part of the SDS–10% polyacrylamide gel with the cellular lysates is shown separately, and an autoradiogram of metabolically labeled purified FV particles separated by SDS-PAGE is shown to the right. (C) Western blot analysis (SDS-PAGE [12% gel]) of cellular lysates and purified FV particles using polyclonal rabbit sera specific for Gag (α-Gag) or SP Env (α-SP). (D) Western blot analysis (SDS-PAGE [12% gel]) of equilibrium sedimentation gradient fractions using polyclonal rabbit sera specific for Gag (α-Gag) and SP Env (α-SP). (E) Pulse-chase analysis of FV Env maturation. Transfected 293T cells were pulse-labeled for 30 min and then chased for different time periods as indicated at the top with fresh growth medium containing an excess of cold methionine and cysteine. Equal cell lysate samples were immunoprecipitated with FV-specific antisera as indicated to the left. Equivalent aliquots of the protein A eluates were incubated with glycosidases as indicated at the top prior to separation by SDS-PAGE (7.5% gel). Identities of the different forms (g [fully glycosylated], h [endo H resistant] and p [N-deglycosylated]) of gp130 Env (solid arrows), gp80 SU (shaded arrows), and gp48 TM (open arrows) are indicated. The bands marked with asterisks at the 1- and 3-h time point after immunoprecipitation with anti-SP/SU antiserum and PNGase F treatment represent the fully N-deglycosylated form of SU running only slightly faster than the fully glycosylated form of TM.

    Article Snippet: For glycosidase treatment, protein A-Sepharose eluates were denatured by boiling in 0.5% SDS–1% β-mercaptoethanol and subsequently incubated with endoglycosidase H (endo H) peptide N -glycosidase or (PNGase F) in the appropriate incubation buffer as suggested by the manufacturer (New England Biolabs) prior to loading on gels for SDS-polyacrylamide gel electrophoresis (PAGE).

    Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Western Blot, Labeling, Metabolic Labelling, Purification, SDS Page, Sedimentation, Pulse Chase, Immunoprecipitation, Incubation

    Implication of CD44 in coat recovery. A , Western blots of CD44 using HCAM antibody with and without endoglycosidase F treatment. B and C , coat thickness measured by the particle exclusion assay. In B , after treatment with 5 units/ml Streptomyces hyaluronidase

    Journal: The Journal of Biological Chemistry

    Article Title: Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions *

    doi: 10.1074/jbc.M109.044362

    Figure Lengend Snippet: Implication of CD44 in coat recovery. A , Western blots of CD44 using HCAM antibody with and without endoglycosidase F treatment. B and C , coat thickness measured by the particle exclusion assay. In B , after treatment with 5 units/ml Streptomyces hyaluronidase

    Article Snippet: In some cases, cell lysates were pretreated with endoglycosidase F (New England Biolabs).

    Techniques: Western Blot, Exclusion Assay

    The pH-responsive Rim101 pathway is involved in lipid asymmetry signaling. (A and B) Cells carrying single or double mutations were grown in YPD medium. Total proteins (1.7 μg) prepared from each culture were incubated with Endo H, separated by

    Journal: Molecular Biology of the Cell

    Article Title: The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry

    doi: 10.1091/mbc.E07-08-0806

    Figure Lengend Snippet: The pH-responsive Rim101 pathway is involved in lipid asymmetry signaling. (A and B) Cells carrying single or double mutations were grown in YPD medium. Total proteins (1.7 μg) prepared from each culture were incubated with Endo H, separated by

    Article Snippet: Proteins (5 μl) in 1× SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and a trace amount of bromophenol blue containing 5% 2-mercaptoethanol) were diluted with 40 μl 0.05 M sodium citrate (pH 5.5) containing 1 mM phenylmethylsulfonyl fluoride, and treated at 37°C for 1 h with 1000 U of recombinant endoglycosidase H (Endo H; New England Biolabs, Beverly, MA).

    Techniques: Incubation