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 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    endoglycosidaseh - by Bioz Stars, 2021-04
    86/100 stars

    Images

    1) Product Images from "CEACAM1 regulates TIM–3–mediated tolerance and exhaustion"

    Article Title: CEACAM1 regulates TIM–3–mediated tolerance and exhaustion

    Journal: Nature

    doi: 10.1038/nature13848

    CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P
    Figure Legend Snippet: CEACAM1 and TIM-3 heterodimerize and serve as heterophilic ligands a, b , Co-immunoprecipitation (IP) and immunoblot (IB) of wild-type hCEACAM1 and hTIM-3 in co-transfected HEK293T cells, c, d , Co-immunoprecipitation and immunoblot of wild-type hCEACAM1 and hTIM-3 mutants ( c ) or wild-type hTIM-3 and hCEACAM1 mutants ( d ) as in a and b. e , Human CEACAM1 (IgV)-TIM-3 (IgV) heterodimer structure, f, g , 2 F o — F c maps contoured at 0.9σ showing electron densities, h, i , Autoradiogram of anti-haemagglutinin (HA) (hTIM-3) immunoprecipitate from metabolic-labelled ( h ) and pulse-chase metabolic-labelled ( i ) co-transfected HEK293T cells. CHO, carbohydrate; core T, non-glycosylated hTIM-3; Cw, wild-type hCEACAM1; EndoH, endoglycosidaseH; H2-MA, HA-tagged influenza virus A M2 protein; T, hTIM-3 (Thr101Ile); Tw, wild-type hTIM-3. hTIM-3 isoforms noted. j , Quantification of densities in i ( n = 3 per group). k , Immunoblot for mTIM-3 from PBS-treated (−) or SEB-treated (+) CD4 + T cells. Labelling as in h and i. 1 , mTIM-3 expression after SEB tolerance induction, m , Column-bound glutathione S -transferase (GST)-hTIM-3 IgV-domain pull-down of hCEACAM1 detected by immunoblot. GST 2 , GST-hTIM-3 dimer. Ft, flow through, n , Suppression of mouse CD4 + T-cell proliferation by mCEACAM1 N-terminal domain-Fc fusion protein (NFc). o , Immunoprecipitation of mTIM-3 and immunoblot for BAT3 or mTIM-3 from lysates of CD4 + T cells. p, q , Proliferation of CD4 + T cells from wild-type ( p ) and CeaCAM1 −/− ( q ) mice transduced with wild-type mTIM-3 (Tw), mTIM-3 Δ252–281 (Tmut) or vector exposed to anti-CD3 and either NFc or IgG1-Fc (IgG1). Data are mean ± s.e.m. and represent five ( a, b ), four ( c, d ), three ( h-j, l, n, p, q ) and two ( k, m, o ) independent experiments. NS, not significant; * P

    Techniques Used: Immunoprecipitation, Transfection, Pulse Chase, Expressing, Flow Cytometry, Mouse Assay, Transduction, Plasmid Preparation

    2) 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

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    Fast Protein Liquid Chromatography:

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    Interaction between ClC-5 and barttin in non-polarizing HEK293T cells. (A) Major expression sites of ClC-5 and ClC-K/barttin reported in the literature (PT, proximal tubule; tDL, thin descending limb of Henle’s loop; tAL and TAL, thin and thick ascending limbs of the Henle’s loop; DCT, distal convoluted tubule; CNT, connecting tubule; CCT, cortical collecting tubule; CD, collecting duct). (B) Alignment showing the sequence conservation of the protein region containing ClC-5 G261E, a Dent disease 1 mutation with Bartter-like phenotype (bold). (C) False-color representation of a fluorescent SDS-PAGE gel of HEK293T cell lysates containing expressed ClC-5-mYFP WT or ClC-5-mYFP G261E. Lysates were incubated with <t>PNGaseF</t> or <t>EndoH</t> to cleave all types or specifically the high mannose N-linked glycosylation, respectively. The resistance of ClC-5 to EndoH indicates complex glycosylation. (D) Representative confocal images of HEK293T cells expressing ClC-5 mCherry or barttin mCFP. Scale bars here and hereafter correspond to 10 μm. (E) Representative confocal image of HEK293T cells expressing ClC-5 G261E mCherry. (F,G) Representative confocal images of HEK293T cells coexpressing barttin (green) together with ClC-5 WT or ClC-5 G261E (ClC-5 in red). Magnified regions of interest are included as insets [in panel (F) , “i” denotes ER staining, whereas “ii” denotes staining of the perinuclear space]. (H) Grayscale presentation of a fluorescent SDS-PAGE gel of HEK293T cell lysates with expressed ClC-5-mCerulean or ClC-5-mCerulean G261E with or without coexpressed barttin mCherry. A brief exposure of intact cells to α-chymotrypsin was used to selectively cleave surface-exposed proteins. (I) Percentage of the low molecular ClC-5 protein band obtained from densitometry analysis of data as depicted in panel (H) , n = 7–11. The intensity of the lower band increases due to cleavage of surface exposed proteins by α-chymotrypsin and is proportional to the PM abundance of the investigated protein. (J) Percentage of the low molecular barttin protein band obtained from densitometry analysis of data as depicted in panel (H) , n = 7–11.
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    Interaction between ClC-5 and barttin in non-polarizing HEK293T cells. (A) Major expression sites of ClC-5 and ClC-K/barttin reported in the literature (PT, proximal tubule; tDL, thin descending limb of Henle’s loop; tAL and TAL, thin and thick ascending limbs of the Henle’s loop; DCT, distal convoluted tubule; CNT, connecting tubule; CCT, cortical collecting tubule; CD, collecting duct). (B) Alignment showing the sequence conservation of the protein region containing ClC-5 G261E, a Dent disease 1 mutation with Bartter-like phenotype (bold). (C) False-color representation of a fluorescent SDS-PAGE gel of HEK293T cell lysates containing expressed ClC-5-mYFP WT or ClC-5-mYFP G261E. Lysates were incubated with PNGaseF or EndoH to cleave all types or specifically the high mannose N-linked glycosylation, respectively. The resistance of ClC-5 to EndoH indicates complex glycosylation. (D) Representative confocal images of HEK293T cells expressing ClC-5 mCherry or barttin mCFP. Scale bars here and hereafter correspond to 10 μm. (E) Representative confocal image of HEK293T cells expressing ClC-5 G261E mCherry. (F,G) Representative confocal images of HEK293T cells coexpressing barttin (green) together with ClC-5 WT or ClC-5 G261E (ClC-5 in red). Magnified regions of interest are included as insets [in panel (F) , “i” denotes ER staining, whereas “ii” denotes staining of the perinuclear space]. (H) Grayscale presentation of a fluorescent SDS-PAGE gel of HEK293T cell lysates with expressed ClC-5-mCerulean or ClC-5-mCerulean G261E with or without coexpressed barttin mCherry. A brief exposure of intact cells to α-chymotrypsin was used to selectively cleave surface-exposed proteins. (I) Percentage of the low molecular ClC-5 protein band obtained from densitometry analysis of data as depicted in panel (H) , n = 7–11. The intensity of the lower band increases due to cleavage of surface exposed proteins by α-chymotrypsin and is proportional to the PM abundance of the investigated protein. (J) Percentage of the low molecular barttin protein band obtained from densitometry analysis of data as depicted in panel (H) , n = 7–11.

    Journal: Frontiers in Physiology

    Article Title: Barttin Regulates the Subcellular Localization and Posttranslational Modification of Human Cl-/H+ Antiporter ClC-5

    doi: 10.3389/fphys.2018.01490

    Figure Lengend Snippet: Interaction between ClC-5 and barttin in non-polarizing HEK293T cells. (A) Major expression sites of ClC-5 and ClC-K/barttin reported in the literature (PT, proximal tubule; tDL, thin descending limb of Henle’s loop; tAL and TAL, thin and thick ascending limbs of the Henle’s loop; DCT, distal convoluted tubule; CNT, connecting tubule; CCT, cortical collecting tubule; CD, collecting duct). (B) Alignment showing the sequence conservation of the protein region containing ClC-5 G261E, a Dent disease 1 mutation with Bartter-like phenotype (bold). (C) False-color representation of a fluorescent SDS-PAGE gel of HEK293T cell lysates containing expressed ClC-5-mYFP WT or ClC-5-mYFP G261E. Lysates were incubated with PNGaseF or EndoH to cleave all types or specifically the high mannose N-linked glycosylation, respectively. The resistance of ClC-5 to EndoH indicates complex glycosylation. (D) Representative confocal images of HEK293T cells expressing ClC-5 mCherry or barttin mCFP. Scale bars here and hereafter correspond to 10 μm. (E) Representative confocal image of HEK293T cells expressing ClC-5 G261E mCherry. (F,G) Representative confocal images of HEK293T cells coexpressing barttin (green) together with ClC-5 WT or ClC-5 G261E (ClC-5 in red). Magnified regions of interest are included as insets [in panel (F) , “i” denotes ER staining, whereas “ii” denotes staining of the perinuclear space]. (H) Grayscale presentation of a fluorescent SDS-PAGE gel of HEK293T cell lysates with expressed ClC-5-mCerulean or ClC-5-mCerulean G261E with or without coexpressed barttin mCherry. A brief exposure of intact cells to α-chymotrypsin was used to selectively cleave surface-exposed proteins. (I) Percentage of the low molecular ClC-5 protein band obtained from densitometry analysis of data as depicted in panel (H) , n = 7–11. The intensity of the lower band increases due to cleavage of surface exposed proteins by α-chymotrypsin and is proportional to the PM abundance of the investigated protein. (J) Percentage of the low molecular barttin protein band obtained from densitometry analysis of data as depicted in panel (H) , n = 7–11.

    Article Snippet: PNGaseF and EndoH (New England Biolabs) were used to determine the type of the ClC-5 glycosylation.

    Techniques: Expressing, Sequencing, Mutagenesis, SDS Page, Incubation, Staining

    D244A and D464A mutants possess no proteolytic activity. (A) SDS-PAGE analysis with or without EndoH treatment. The D244A and D464A mutants prevented fragmentation, and lost the 15-kDa fragment found in the Sap7 wild type lane. M: marker. (B) Proteolytic activity of the mutants. The D244A and D464A mutants completely lost their proteolytic activity. This result suggests that D244 and D464 represent the active site of Sap7. Averages of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: D244A and D464A mutants possess no proteolytic activity. (A) SDS-PAGE analysis with or without EndoH treatment. The D244A and D464A mutants prevented fragmentation, and lost the 15-kDa fragment found in the Sap7 wild type lane. M: marker. (B) Proteolytic activity of the mutants. The D244A and D464A mutants completely lost their proteolytic activity. This result suggests that D244 and D464 represent the active site of Sap7. Averages of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

    Article Snippet: SDS-PAGE, CBB staining, and western blotting The purified Sap isozymes were separated by SDS-PAGE with or without EndoH (New England Biolabs, MA, USA) treatment in a 5%–20% gradient polyacrylamide gel.

    Techniques: Activity Assay, SDS Page, Marker

    N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: N -glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A. (A) Proteolytic activity of deglycosylated Sap7. The influence of N -glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments ( Fig. 1A ) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.

    Article Snippet: SDS-PAGE, CBB staining, and western blotting The purified Sap isozymes were separated by SDS-PAGE with or without EndoH (New England Biolabs, MA, USA) treatment in a 5%–20% gradient polyacrylamide gel.

    Techniques: Activity Assay, SDS Page, Marker

    M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site. (A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by P. pastoris , and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** P

    Article Snippet: SDS-PAGE, CBB staining, and western blotting The purified Sap isozymes were separated by SDS-PAGE with or without EndoH (New England Biolabs, MA, USA) treatment in a 5%–20% gradient polyacrylamide gel.

    Techniques: SDS Page, Produced, Marker, Activity Assay, Mutagenesis

    Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

    Journal: PLoS ONE

    Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

    doi: 10.1371/journal.pone.0032513

    Figure Lengend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

    Article Snippet: SDS-PAGE, CBB staining, and western blotting The purified Sap isozymes were separated by SDS-PAGE with or without EndoH (New England Biolabs, MA, USA) treatment in a 5%–20% gradient polyacrylamide gel.

    Techniques: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

    Effect of single N-glycosylation site removal on the cell surface levels and stability of human TpoR . (A) Sorted Ba/F3 parental or Ba/F3-JAK2 cells, expressing TpoR variants defective in one N-glycosylation site [Δ (1), Δ (2), Δ (3), Δ (4)] were tested for cell surface levels using anti-HA antibody based flow cytometry. Similar results were obtained in four independent experiments, on two different cell lines obtained for each TpoR variant. The differences between the cell surface levels of Δ (1) TpoR in Ba/F3 and Ba/F3-JAK2 cells were statistically significant when compared with the levels of WT TpoR ( p = 0.006 and p = 0.0086, respectively). (B) HEK293 cells were plated in 6-well plates and transiently transfected with 3 μg of each of the indicated TpoR variants. Twenty-four hours post-transfection cells were lysed and treated with EndoH or PNGaseF or left untreated and subjected to Western blot analysis. Membranes were probed with anti-HA. Similar results were obtained in three different experiments. (C) A pool of the cell lines used in (A) were treated at 37°C with 50 μg/ml cycloheximide (CHX) over different time periods then lysed. The lysates were analyzed for TpoR variants stability using anti-HA and normalizing by anti-β-actin Western blotting.

    Journal: Frontiers in Endocrinology

    Article Title: Extracellular Domain N-Glycosylation Controls Human Thrombopoietin Receptor Cell Surface Levels

    doi: 10.3389/fendo.2011.00071

    Figure Lengend Snippet: Effect of single N-glycosylation site removal on the cell surface levels and stability of human TpoR . (A) Sorted Ba/F3 parental or Ba/F3-JAK2 cells, expressing TpoR variants defective in one N-glycosylation site [Δ (1), Δ (2), Δ (3), Δ (4)] were tested for cell surface levels using anti-HA antibody based flow cytometry. Similar results were obtained in four independent experiments, on two different cell lines obtained for each TpoR variant. The differences between the cell surface levels of Δ (1) TpoR in Ba/F3 and Ba/F3-JAK2 cells were statistically significant when compared with the levels of WT TpoR ( p = 0.006 and p = 0.0086, respectively). (B) HEK293 cells were plated in 6-well plates and transiently transfected with 3 μg of each of the indicated TpoR variants. Twenty-four hours post-transfection cells were lysed and treated with EndoH or PNGaseF or left untreated and subjected to Western blot analysis. Membranes were probed with anti-HA. Similar results were obtained in three different experiments. (C) A pool of the cell lines used in (A) were treated at 37°C with 50 μg/ml cycloheximide (CHX) over different time periods then lysed. The lysates were analyzed for TpoR variants stability using anti-HA and normalizing by anti-β-actin Western blotting.

    Article Snippet: Cell lysates were digested with EndoH or PNGaseF (both from New England BioLabs) or left untreated for 16 h at 37°C.

    Techniques: Expressing, Flow Cytometry, Cytometry, Variant Assay, Transfection, Western Blot

    Effect of multiple N-glycosylation sites removal on the cell surface levels and function of human TpoR . (A) Sorted Ba/F3 parental or Ba/F3-JAK2 cells, expressing TpoR variants defective in three N-glycosylation sites [Δ (123), Δ (124), Δ (134), Δ (234)] were tested for cell surface levels using anti-HA antibody based flow cytometry. Similar results were obtained in two independent experiments, on two different cell lines obtained for each TpoR variant. For the Ba/F3 cells, the t -test showed statistically significant differences between Δ (123) TpoR ( p = 0.0019), Δ (124) TpoR ( p = 0.0036), Δ (134) TpoR ( p = 0.0016), and Δ (234) TpoR ( p = 0.0086) when compared with WT TpoR. (B) HEK293 cells were seeded in 6-well plates and transiently transfected with 3 μg of each of the indicated TpoR variants. Twenty-four hours post-transfection cells were lysed and treated with EndoH or PNGaseF or left untreated during 16 h at 37°C. The digestion products were subjected to Western blot analysis. Membranes were probed with anti-HA and anti-β-actin. Similar results were obtained in two different experiments. (C) A pool of each cell line used in (A) was treated at 37°C with 50 μg/ml cycloheximide (CHX) over different time periods then lysed. The lysates were analyzed for TpoR variants stability using anti-HA and normalized by anti-β-actin Western blotting. (D) Ba/F3 or Ba/F3-JAK2 cells stably expressing each human TpoR defective in three N-glycosylation sites were starved 3 h in RPMI medium +1 mg/ml BSA and electroporated with the pGRR5-luc and pRLTK-luc reporters. The cells were stimulated with 50 ng Tpo/ml or left unstimulated 2 h at 37°C. Upon treatment, the cells were lysed and their luminescence recorded. Results are the mean ± variation of triplicate samples. One of three independent experiments is depicted. The t -test showed statistically significant differences for Tpo-dependent transcriptional activity levels of the all the TpoR variants, when compared with the WT TpoR (all the p values

    Journal: Frontiers in Endocrinology

    Article Title: Extracellular Domain N-Glycosylation Controls Human Thrombopoietin Receptor Cell Surface Levels

    doi: 10.3389/fendo.2011.00071

    Figure Lengend Snippet: Effect of multiple N-glycosylation sites removal on the cell surface levels and function of human TpoR . (A) Sorted Ba/F3 parental or Ba/F3-JAK2 cells, expressing TpoR variants defective in three N-glycosylation sites [Δ (123), Δ (124), Δ (134), Δ (234)] were tested for cell surface levels using anti-HA antibody based flow cytometry. Similar results were obtained in two independent experiments, on two different cell lines obtained for each TpoR variant. For the Ba/F3 cells, the t -test showed statistically significant differences between Δ (123) TpoR ( p = 0.0019), Δ (124) TpoR ( p = 0.0036), Δ (134) TpoR ( p = 0.0016), and Δ (234) TpoR ( p = 0.0086) when compared with WT TpoR. (B) HEK293 cells were seeded in 6-well plates and transiently transfected with 3 μg of each of the indicated TpoR variants. Twenty-four hours post-transfection cells were lysed and treated with EndoH or PNGaseF or left untreated during 16 h at 37°C. The digestion products were subjected to Western blot analysis. Membranes were probed with anti-HA and anti-β-actin. Similar results were obtained in two different experiments. (C) A pool of each cell line used in (A) was treated at 37°C with 50 μg/ml cycloheximide (CHX) over different time periods then lysed. The lysates were analyzed for TpoR variants stability using anti-HA and normalized by anti-β-actin Western blotting. (D) Ba/F3 or Ba/F3-JAK2 cells stably expressing each human TpoR defective in three N-glycosylation sites were starved 3 h in RPMI medium +1 mg/ml BSA and electroporated with the pGRR5-luc and pRLTK-luc reporters. The cells were stimulated with 50 ng Tpo/ml or left unstimulated 2 h at 37°C. Upon treatment, the cells were lysed and their luminescence recorded. Results are the mean ± variation of triplicate samples. One of three independent experiments is depicted. The t -test showed statistically significant differences for Tpo-dependent transcriptional activity levels of the all the TpoR variants, when compared with the WT TpoR (all the p values

    Article Snippet: Cell lysates were digested with EndoH or PNGaseF (both from New England BioLabs) or left untreated for 16 h at 37°C.

    Techniques: Expressing, Flow Cytometry, Cytometry, Variant Assay, Transfection, Western Blot, Stable Transfection, Activity Assay

    Mutations in IGSF1 impair its plasma membrane trafficking (a) HEK293 cells were transfected with pcDNA3 (empty vector) or the indicated wild-type or mutant IGSF1 expression vectors. Protein lysates were deglycosylated with either PNGaseF (P) or EndoH (E), resolved by SDS-PAGE, and immunoblotted using an IGSF1-CTD antibody. Non-specific bands are indicated by *. (b) HEK293 cells were transfected with the same constructs as in (a). Expression of IGSF1-CTD was analyzed by immunofluorescence using the IGSF1-CTD antibody under non-permeabilizing and permeabilizing conditions. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. (c) HEK293 cells were transfected with pcDNA3 or the indicated wild-type or mutant IGSF1 expression vectors. Membrane expression of IGSF1-CTD was analyzed by cell-surface biotinylation.

    Journal: Nature genetics

    Article Title: Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement

    doi: 10.1038/ng.2453

    Figure Lengend Snippet: Mutations in IGSF1 impair its plasma membrane trafficking (a) HEK293 cells were transfected with pcDNA3 (empty vector) or the indicated wild-type or mutant IGSF1 expression vectors. Protein lysates were deglycosylated with either PNGaseF (P) or EndoH (E), resolved by SDS-PAGE, and immunoblotted using an IGSF1-CTD antibody. Non-specific bands are indicated by *. (b) HEK293 cells were transfected with the same constructs as in (a). Expression of IGSF1-CTD was analyzed by immunofluorescence using the IGSF1-CTD antibody under non-permeabilizing and permeabilizing conditions. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. (c) HEK293 cells were transfected with pcDNA3 or the indicated wild-type or mutant IGSF1 expression vectors. Membrane expression of IGSF1-CTD was analyzed by cell-surface biotinylation.

    Article Snippet: Protein lysates were deglycosylated using PNGaseF and EndoH (New England Biolabs), using manufacturer’s instructions.

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing, SDS Page, Construct, Immunofluorescence, Staining