β mercaptoethanol  (Thermo Fisher)


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    Name:
    Gibco 2 Mercaptoethanol
    Description:
    Gibco 2 Mercaptoethanol also known as beta mercaptoethanol or BME is a potent reducing agent used in cell culture media to prevent toxic levels of oxygen radicals 2 Mercaptoethanol is not stable in solution so most protocols require daily supplementation Gibco 2 Mercaptoethanol contains 2 mercaptoethanol at a concentration of 55 mM in Dulbecco s phosphate buffered saline DPBS cGMP manufacturing and quality systemGibco Reducing Agent is manufactured at a cGMP compliant facility located in Grand Island New York The facility is registered with the FDA as a medical device manufacturer and is certified to ISO 13485 standards
    Catalog Number:
    21985023
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Mammalian Cell Culture
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    Structured Review

    Thermo Fisher β mercaptoethanol
    Gibco 2 Mercaptoethanol also known as beta mercaptoethanol or BME is a potent reducing agent used in cell culture media to prevent toxic levels of oxygen radicals 2 Mercaptoethanol is not stable in solution so most protocols require daily supplementation Gibco 2 Mercaptoethanol contains 2 mercaptoethanol at a concentration of 55 mM in Dulbecco s phosphate buffered saline DPBS cGMP manufacturing and quality systemGibco Reducing Agent is manufactured at a cGMP compliant facility located in Grand Island New York The facility is registered with the FDA as a medical device manufacturer and is certified to ISO 13485 standards
    https://www.bioz.com/result/β mercaptoethanol/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    β mercaptoethanol - by Bioz Stars, 2021-03
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    Related Articles

    Electrophoresis:

    Article Title: Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein
    Article Snippet: After addition of 75 µl Dual-Glo Luciferase Reagent (Promega) to each well, the plate was incubated for 10 min. Luminescence was measured using a SpectraMax M5 microplate reader (Molecular Devices). .. Proteins were treated with SDS and β-mercaptoethanol at 95 °C, resolved by electrophoresis on Novex Tris-glycine gels (Invitrogen) and transferred onto nitrocellulose membranes. .. Afterwards, the membranes were blocked with PBS containing 5 % Carnation powdered milk and 0.1 % Tween-20 and then incubated with guinea pig VP6 or NSP3 antiserum in PBS containing 1 % milk and 0.1 % Tween-20.

    Incubation:

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms
    Article Snippet: Aliquots of CHO-K1 cell lysates, stably overexpressing APP constructs, were either incubated with SDS sample loading buffer (0.625 M Tris-HCl, pH 6.8, 2% w/v SDS, 10% w/v glycerol, 5% β-mercaptoethanol) and directly subjected to SDS-PAGE, or heated at indicated temperatures. .. Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). .. Non-specific binding to membranes was blocked 1 h with 5% non-fat dry milk in TBS containing 0.01% Tween-20 (Roth, Germany), before incubation with the appropriate primary and secondary antibodies.

    Cell Culture:

    Article Title: The NS1 Protein of Influenza A Virus Participates in Necroptosis by Interacting with MLKL and Increasing Its Oligomerization and Membrane Translocation
    Article Snippet: Human embryonic kidney HEK293T and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Sigma). .. THP1 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 0.05 mM β-mercaptoethanol (Gibco). .. Phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) was added to the THP1 culture (100 ng/ml) at least overnight to differentiate THP1 cells into macrophage-like cells.

    other:

    Article Title: Uncyclized xanthommatin is a key ommochrome intermediate in invertebrate coloration
    Article Snippet: Sucrose (99 %) and sulfurous acid (6 % SO2) were purchased from Alfa Aesar. β-Mercaptoethanol was purchased from BDH Chemicals.

    Article Title: Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-Terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos
    Article Snippet: Three aliquots of 10 μL of each sample were taken and treated with (1) 5 μL of loading buffer with β-mercaptoethanol, followed by 5 min of heating at 95 °C; (2) 5 μL of loading buffer with 50 mM TCEP; and (3) 5 μL of loading buffer.

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    Thermo Fisher β mercaptoethanol
    ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing <t>β-mercaptoethanol</t> (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA
    β Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher ins1e cells
    NNAT interaction with the SPC and modulation of preproinsulin handling. ( A ) Overview of affinity purification/mass spectrometry (AP/MS) screen for novel interaction partners of NNAT. Endogenous NNAT was immunoprecipitated (IP) from MIN6 cell lysates and interacting partners in co-IPs analyzed by liquid chromatography/mass spectrometry (LC/MS). ( B ) Heatmap from AP/MS analysis of top protein hits in IPs using antibodies against NNAT (NNAT Ab) and control IPs with rabbit immunoglobulins (IgG). Relatively high abundance is shown in yellow and relatively low abundance in blue. ( C ) Lysates from HEK293T cells expressing c-Myc–tagged SEC11A and FLAG-tagged NNAT were immunoprecipitated using anti–c-Myc antibodies. Proteins in input and IP samples were detected by Western blotting using anti–c-Myc and anti-FLAG antibodies. Panel shows a representative blot of 3 independent experiments. ( D ) <t>INS1E</t> cells transiently transfected with siRNA targeting Nnat or Sec11a were assayed in vitro for GSIS at low (3 mM) and high (25 mM) glucose. A scramble siRNA served as a control with data expressed as mean insulin secretion per unit cellular protein. Graph on the right shows total insulin content in cell lysates. ( n = 9 independent cultures per group, 3 independent experiments, 2-way ANOVA [left graph] and 1-way ANOVA [right graph].) ( E ) INS1E cells transfected with c-Myc–tagged preproinsulin and siRNAs targeting Nnat or Sec11a were pulse-labeled with 35 S-Cys/Met. Lysates immunoprecipitated with anti–c-Myc agarose were analyzed by autoradiography. Associated bar chart shows preproinsulin and proinsulin band intensities in multiple experiments quantified by densitometry and expressed as percentage processing of preproinsulin ( n = 3 cultures per group from 3 independent experiments, 1-way ANOVA). ( F ) Similar experiments performed as in E , from 3-hour chase cell lysates (C) and media (M), quantified as in E ( n = 4 cultures per group from 3 independent experiments, 1-way ANOVA for both C and M). (* P
    Ins1e Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher smln cultured cells
    Effect of <t>N-PmpC</t> and CtB stimulation on the in vitro cytokine pattern in <t>SMLN</t> cells isolated after ocular mucosal immunization with N-PmpC and N-PmpC/LB. Bar graphs representing the levels of IFNγ, IL-4, IL-10 and IL-17A in the supernatants of non-stimulated, N-PmpC- or CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva (bars). The corresponding measurements in SMLN cells isolated from age-matched controls (nc) are presented on the graphs as a solid line (mean value) and dotted lines (upper and lower standard error values). SMLN cells were cultured at 37°C under a 5% CO 2 atmosphere for 48 h in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10 6 CFU/ml for CtB). The results are presented as the mean concentrations ± SE (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc stimulated in a same way P
    Smln Cultured Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing β-mercaptoethanol (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA

    Journal: Cellular and Molecular Life Sciences

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    doi: 10.1007/s00018-011-0882-4

    Figure Lengend Snippet: ER-retention of APP by addition of the di-lysine motif KKAA induces SDS stable homo-dimers in an oxidizing cellular surrounding. a Schematic illustration of the APP domain structure and the used dilysine retention constructs. APP isoform 695 (APP695) was fused to a C-terminal myc-tag, followed by either a KKAA ER retention motif, or a “leaky” KKFF motif allowing trafficking to the cis -Golgi compartment. The E1 domain, acidic domain, E2 domain, transmembrane domain (TM), and the intracellular domain are indicated. b CHO-K1 cells stably over expressing the respective APP695 wt or APP695 retention constructs were subjected to cell surface biotinylation. To inhibit endocytosis from the cell surface, AP180 D/N (lane four) was transiently co-expressed. At 24-h post-transfection, cell surface proteins were labeled with sulfo NHS-SS-biotin at 4°C, prior to lysis. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and Western blot (PD: NeutrAvidin; right panel ). As input controls, 20 μg of cell lysates were used (input; left panel ). APP full length, CTFs and AP180 D/N were probed with 9E10 antibody. Monoclonal α-Tubulin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. For the input control, all samples were treated with SDS sample loading buffer, containing β-mercaptoethanol (BME) without heat denaturing prior to SDS gel electrophoresis. To elute surface proteins from NeutrAvidin beads, samples were denatured for 5 min at 95°C with BME and separated on 4–12% SDS gels. Molecular weight standards (kDa) are indicated on the left . c Cell lysates (20 μg each lane) of CHO-K1 cells stably overexpressing APP ER (KKAA), were incubated with SDS sample loading buffer (+ BME) and heat denatured for 10 min at the indicated temperatures (4–85°C). APP monomers and SDS stable dimers were detected, using the monoclonal anti myc antibody (9E10), shown on one representative Western blot. d APP dimers and monomers were quantified by densitometric analysis of Western blots, as the one shown in c . In each case, intensities at 4°C were set as 100%. Diagram shows relative intensity (%) of APP monomers and dimers, plotted against different temperatures. Data are ( n ≥ 3) ± SEM, one-way ANOVA

    Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).

    Techniques: Construct, Stable Transfection, Expressing, Transfection, Labeling, Lysis, SDS Page, Western Blot, SDS-Gel, Electrophoresis, Molecular Weight, Incubation

    APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Journal: Cellular and Molecular Life Sciences

    Article Title: APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

    doi: 10.1007/s00018-011-0882-4

    Figure Lengend Snippet: APP wt homodimers are formed in the ER but also occur at the cell surface. a N2a cells were transiently co-transfected with NT HA APP695 CT Split GFP 1–10 and NT HA APP695 CT Split GFP 11. The cells were fixed with paraformaldehyde and immunostained with ER marker Grp78, followed by incubation with AlexaFlour-594 conjugated secondary antibody. The green fluorescence represents the APP dimer, induced by bimolecular fluorescence complementation (BiFC). Co-staining with Grp78 revealed colocalization of the APP dimer ( inset ) with the ER. b Single transfections of N2a cells with either NT HA APP695 CT Split GFP 1–10 or NT HA APP695 CT Split GFP 11. APP expression was verified with HA antibody, confirming a non-perturbed distribution ( right panel , red ). The green channel does not show any specific fluorescence. Scale bar is 10 μm. c N2a cells were transiently transfected with NT HA APP695 CT Split GFP 1–10, NT HA APP695 CT Split GFP 11 or GFP only. Cell lysates were analyzed via Western blotting for APP full length ( top part ), APP CTFs ( middle part ) and GFP, or GFP-containing CTFs ( bottom part ). Note that GFP antibody does not recognize GFP 11 alone. d CHO-K1 cells stably overexpressing APP 695 wt were pretreated for 1 h at 37°C in the presence of 10 μg/ml brefeldin A (BFA), or DMSO (vehicle) for 1 h to block protein secretion from the ER. Subsequently, cells were either directly lysed (BFA), or medium containing BFA was removed, and cells were incubated for additional 30 min at 37°C with 10 μM chlorpromazine (BFA/CPZ), to allow transport to the cell surface, but inhibit further endocytosis. Lysates were analyzed either under non-reducing (+β mercaptoethanol; −95°C), or reducing conditions (+β-mercaptoethanol; +95°C). Note that cells pretreated with BFA, followed by chlorpromazine exposure (BFA/CPZ), display two upper SDS-resistant bands migrating with a slight size shift, indicating different glycosylation patterns (dimer i/m). Note that strong denaturing conditions (+95°C) result in the disappearance of the upper migrating bands, suggesting disulfide bond formation. The separating line indicates different exposure times to visualize dimer formation in treated cells, whereas APP ER overexpressing cells served as size reference for dimers. e Cells were treated as described under d , and subjected to cell surface biotinylation to confirm drug treatments. APP ER is completely absent from the surface, whereas only minor quantities of mature APP reach the surface upon BFA treatment. Removal of BFA, and subsequent incubation with chlorpromazine (BFA/CPZ), strongly increases surface exposed mature APP, compared to vehicle control. Note that no dimers are recovered in biotinylation, because NeutrAvidin beads were boiled in BME containing sample buffer, to release biotin conjugated proteins

    Article Snippet: Alternatively, samples were incubated with sample buffer without β-mercaptoethanol and heat denatured for 10 min. Proteins were electrophoresed on 4–12% NuPage (Novex®, Invitrogen) gradient gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA).

    Techniques: Transfection, Marker, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Staining, Expressing, Western Blot, Stable Transfection, Blocking Assay

    Disruption of ICJ in respiratory mucosal explants. ( a ) The percentage of intercellular spaces in equine respiratory mucosal explants after 1 h treatment with PBS (control), DTT, β-mercaptoethanol, NAC or EGTA (left) and 24 h after the 1 h treatment (right). Three independent experiments were performed and data are represented as means + SD, different lower case letters indicate significant (P

    Journal: Scientific Reports

    Article Title: Access to a main alphaherpesvirus receptor, located basolaterally in the respiratory epithelium, is masked by intercellular junctions

    doi: 10.1038/s41598-017-16804-5

    Figure Lengend Snippet: Disruption of ICJ in respiratory mucosal explants. ( a ) The percentage of intercellular spaces in equine respiratory mucosal explants after 1 h treatment with PBS (control), DTT, β-mercaptoethanol, NAC or EGTA (left) and 24 h after the 1 h treatment (right). Three independent experiments were performed and data are represented as means + SD, different lower case letters indicate significant (P

    Article Snippet: Next, the apical surface of the epithelium was exposed for 1 h at 37 °C to 8 mM ethylene glycol tetra-acetic acid (EGTA) (VWR International, Leuven, Belgium), 500 mM N-acetylcysteine (NAC) (Sigma-Aldrich), 20 mM dithiotreitol (DTT) (Sigma-Aldrich) or 50 mM β-mercaptoethanol (Invitrogen) in PBS.

    Techniques:

    NNAT interaction with the SPC and modulation of preproinsulin handling. ( A ) Overview of affinity purification/mass spectrometry (AP/MS) screen for novel interaction partners of NNAT. Endogenous NNAT was immunoprecipitated (IP) from MIN6 cell lysates and interacting partners in co-IPs analyzed by liquid chromatography/mass spectrometry (LC/MS). ( B ) Heatmap from AP/MS analysis of top protein hits in IPs using antibodies against NNAT (NNAT Ab) and control IPs with rabbit immunoglobulins (IgG). Relatively high abundance is shown in yellow and relatively low abundance in blue. ( C ) Lysates from HEK293T cells expressing c-Myc–tagged SEC11A and FLAG-tagged NNAT were immunoprecipitated using anti–c-Myc antibodies. Proteins in input and IP samples were detected by Western blotting using anti–c-Myc and anti-FLAG antibodies. Panel shows a representative blot of 3 independent experiments. ( D ) INS1E cells transiently transfected with siRNA targeting Nnat or Sec11a were assayed in vitro for GSIS at low (3 mM) and high (25 mM) glucose. A scramble siRNA served as a control with data expressed as mean insulin secretion per unit cellular protein. Graph on the right shows total insulin content in cell lysates. ( n = 9 independent cultures per group, 3 independent experiments, 2-way ANOVA [left graph] and 1-way ANOVA [right graph].) ( E ) INS1E cells transfected with c-Myc–tagged preproinsulin and siRNAs targeting Nnat or Sec11a were pulse-labeled with 35 S-Cys/Met. Lysates immunoprecipitated with anti–c-Myc agarose were analyzed by autoradiography. Associated bar chart shows preproinsulin and proinsulin band intensities in multiple experiments quantified by densitometry and expressed as percentage processing of preproinsulin ( n = 3 cultures per group from 3 independent experiments, 1-way ANOVA). ( F ) Similar experiments performed as in E , from 3-hour chase cell lysates (C) and media (M), quantified as in E ( n = 4 cultures per group from 3 independent experiments, 1-way ANOVA for both C and M). (* P

    Journal: The Journal of Clinical Investigation

    Article Title: Neuronatin regulates pancreatic β cell insulin content and secretion

    doi: 10.1172/JCI120115

    Figure Lengend Snippet: NNAT interaction with the SPC and modulation of preproinsulin handling. ( A ) Overview of affinity purification/mass spectrometry (AP/MS) screen for novel interaction partners of NNAT. Endogenous NNAT was immunoprecipitated (IP) from MIN6 cell lysates and interacting partners in co-IPs analyzed by liquid chromatography/mass spectrometry (LC/MS). ( B ) Heatmap from AP/MS analysis of top protein hits in IPs using antibodies against NNAT (NNAT Ab) and control IPs with rabbit immunoglobulins (IgG). Relatively high abundance is shown in yellow and relatively low abundance in blue. ( C ) Lysates from HEK293T cells expressing c-Myc–tagged SEC11A and FLAG-tagged NNAT were immunoprecipitated using anti–c-Myc antibodies. Proteins in input and IP samples were detected by Western blotting using anti–c-Myc and anti-FLAG antibodies. Panel shows a representative blot of 3 independent experiments. ( D ) INS1E cells transiently transfected with siRNA targeting Nnat or Sec11a were assayed in vitro for GSIS at low (3 mM) and high (25 mM) glucose. A scramble siRNA served as a control with data expressed as mean insulin secretion per unit cellular protein. Graph on the right shows total insulin content in cell lysates. ( n = 9 independent cultures per group, 3 independent experiments, 2-way ANOVA [left graph] and 1-way ANOVA [right graph].) ( E ) INS1E cells transfected with c-Myc–tagged preproinsulin and siRNAs targeting Nnat or Sec11a were pulse-labeled with 35 S-Cys/Met. Lysates immunoprecipitated with anti–c-Myc agarose were analyzed by autoradiography. Associated bar chart shows preproinsulin and proinsulin band intensities in multiple experiments quantified by densitometry and expressed as percentage processing of preproinsulin ( n = 3 cultures per group from 3 independent experiments, 1-way ANOVA). ( F ) Similar experiments performed as in E , from 3-hour chase cell lysates (C) and media (M), quantified as in E ( n = 4 cultures per group from 3 independent experiments, 1-way ANOVA for both C and M). (* P

    Article Snippet: For siRNA silencing, INS1E cells ( ) (cultured in RPMI, 10% FBS, 2 mM l -glutamine, 50 μM β-mercaptoethanol, 37°C, 5% CO2 ) were transfected with 50 nM of Silencer Select siRNAs ( Nnat , s137247; Sec11a , s80747; both Ambion) using Dharmafect 1 reagent (Dharmacon).

    Techniques: Affinity Purification, Mass Spectrometry, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Transfection, In Vitro, Labeling, Autoradiography

    Effect of N-PmpC and CtB stimulation on the in vitro cytokine pattern in SMLN cells isolated after ocular mucosal immunization with N-PmpC and N-PmpC/LB. Bar graphs representing the levels of IFNγ, IL-4, IL-10 and IL-17A in the supernatants of non-stimulated, N-PmpC- or CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva (bars). The corresponding measurements in SMLN cells isolated from age-matched controls (nc) are presented on the graphs as a solid line (mean value) and dotted lines (upper and lower standard error values). SMLN cells were cultured at 37°C under a 5% CO 2 atmosphere for 48 h in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10 6 CFU/ml for CtB). The results are presented as the mean concentrations ± SE (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc stimulated in a same way P

    Journal: PLoS ONE

    Article Title: A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C

    doi: 10.1371/journal.pone.0157875

    Figure Lengend Snippet: Effect of N-PmpC and CtB stimulation on the in vitro cytokine pattern in SMLN cells isolated after ocular mucosal immunization with N-PmpC and N-PmpC/LB. Bar graphs representing the levels of IFNγ, IL-4, IL-10 and IL-17A in the supernatants of non-stimulated, N-PmpC- or CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva (bars). The corresponding measurements in SMLN cells isolated from age-matched controls (nc) are presented on the graphs as a solid line (mean value) and dotted lines (upper and lower standard error values). SMLN cells were cultured at 37°C under a 5% CO 2 atmosphere for 48 h in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10 6 CFU/ml for CtB). The results are presented as the mean concentrations ± SE (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc stimulated in a same way P

    Article Snippet: SMLN cell cytokine profiling Production of IFN-γ, IL-4, IL-17A and IL-10 was analysed by measuring their concentrations in the supernatants of non-stimulated, N-PmpC- (10 μg/ml) and CtB-stimulated (1 x 106 IFU/ml) SMLN cultured cells (2 x 106 cell/ml in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640; 37°C, 5% CO2 , 48 h) using sandwich ELISA with commercially available monoclonal antibodies (eBioscience) [ ].

    Techniques: CtB Assay, In Vitro, Isolation, Mouse Assay, Cell Culture

    In vitro analysis of proliferation of primary SMLN cells subjected to N-PmpC and CtB stimulation. Bar graph showing the proliferation indices (PI) of N-PmpC- and CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva. The proliferation indices of SMLN cells isolated from age-matched control mice (nc) are presented on the graph as a solid line (mean value) and dotted lines (upper and lower standard error values). The number of viable SMLN cells was assessed using the Cell Counting Kit - 8 following a 48 h culture period in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10 6 CFU/ml for CtB). PIs were calculated for each mouse. The results are presented as the mean PIs ± SE for each experimental group (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc P

    Journal: PLoS ONE

    Article Title: A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C

    doi: 10.1371/journal.pone.0157875

    Figure Lengend Snippet: In vitro analysis of proliferation of primary SMLN cells subjected to N-PmpC and CtB stimulation. Bar graph showing the proliferation indices (PI) of N-PmpC- and CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva. The proliferation indices of SMLN cells isolated from age-matched control mice (nc) are presented on the graph as a solid line (mean value) and dotted lines (upper and lower standard error values). The number of viable SMLN cells was assessed using the Cell Counting Kit - 8 following a 48 h culture period in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10 6 CFU/ml for CtB). PIs were calculated for each mouse. The results are presented as the mean PIs ± SE for each experimental group (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc P

    Article Snippet: SMLN cell cytokine profiling Production of IFN-γ, IL-4, IL-17A and IL-10 was analysed by measuring their concentrations in the supernatants of non-stimulated, N-PmpC- (10 μg/ml) and CtB-stimulated (1 x 106 IFU/ml) SMLN cultured cells (2 x 106 cell/ml in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640; 37°C, 5% CO2 , 48 h) using sandwich ELISA with commercially available monoclonal antibodies (eBioscience) [ ].

    Techniques: In Vitro, CtB Assay, Isolation, Mouse Assay, Cell Counting