protease inhibitor mixture  (Roche)


Bioz Verified Symbol Roche is a verified supplier
Bioz Manufacturer Symbol Roche manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Roche protease inhibitor mixture
    Protease Inhibitor Mixture, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease inhibitor mixture/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mixture - by Bioz Stars, 2021-07
    86/100 stars

    Images

    Related Articles

    Expressing:

    Article Title: Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation
    Article Snippet: .. For this protocol, 107 THP-1 cells expressing wild-type or mutant CD1d were lysed for 30 min on ice in 2 mL 0.5% Triton X 100 lysis buffer containing protease inhibitors mixture (Roche). .. Cell debris was removed by 10 min at 15,000 × g 4 °C centrifugation and supernatant was split into several tubes.

    Mutagenesis:

    Article Title: Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation
    Article Snippet: .. For this protocol, 107 THP-1 cells expressing wild-type or mutant CD1d were lysed for 30 min on ice in 2 mL 0.5% Triton X 100 lysis buffer containing protease inhibitors mixture (Roche). .. Cell debris was removed by 10 min at 15,000 × g 4 °C centrifugation and supernatant was split into several tubes.

    Lysis:

    Article Title: Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation
    Article Snippet: .. For this protocol, 107 THP-1 cells expressing wild-type or mutant CD1d were lysed for 30 min on ice in 2 mL 0.5% Triton X 100 lysis buffer containing protease inhibitors mixture (Roche). .. Cell debris was removed by 10 min at 15,000 × g 4 °C centrifugation and supernatant was split into several tubes.

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Activity Assay:

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Protease Inhibitor:

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Article Title: Characterization of Chromoshadow Domain-mediated Binding of Heterochromatin Protein 1? (HP1?) to Histone H3 *
    Article Snippet: 15 N-Labeled CSDα(109–185) was purified on Mono Q and then Superdex 75 gel filtration columns (GE Healthcare). .. GST Pulldown Assays Approximately 5 μg of protein was mixed with a GST fusion protein immobilized on glutathione-Sepharose beads in 250 μl of binding buffer (150 mm NaCl, 20 mm Tris-HCl (pH 7.5), 5 mm DTT, 0.25% Nonidet P-40, and 1× complete protease inhibitor mixture (Roche Applied Science)). .. After incubation at 4 °C for 2 h, the beads were washed four times with 0.7 ml of binding buffer.

    Article Title: The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding
    Article Snippet: .. Western blot analysis For western blot analyses, pellets of infected FHK cells or RK/gH1 were resuspended in radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Roche, Mannheim, Germany). .. Sample buffer (1M Tris/HCl, pH 6.8; 0.8% sodium dodecyl sulfate (SDS); 0.4% glycerol; 0.15% β-mercaptoethanol; 0.004% bromophenol blue) was added, the mixture was heated at 95°C for 5 min and proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) exactly as previously described [ ].

    Article Title: Apical and Basal Matrix Remodeling Control Epithelial Morphogenesis
    Article Snippet: .. Protease Inhibitor Treatment dp-YFP ,nub-Gal4>Timp and vkg-YFP ,nub-Gal4>Timp wing imaginal discs were dissected from the puparium at 4 hours APF and transferred to supplemented Shield and Sang medium including a protease inhibitors mixture (2ml of media containing 1 tablet of cOmplete Protease Inhibitor Cocktail, 04693116001, Roche). .. Wing disc were incubated for 3 hours and 30 mminutes at 25 C, fixed and immunostained.

    Article Title: Membrane Labeling of Coral Gastrodermal Cells by Biotinylation: The Proteomic Identification of Surface Proteins Involving Cnidaria-Dinoflagellate Endosymbiosis
    Article Snippet: After complete drying, grids were observed with a JEM-1400 transmission electron microscope (JEOL, Japan). .. 2D SDS-PAGE analysis of biotinylated proteins Biotinylated SGCs were prepared as described above and suspended in 550 µL modified isotonic RadioImmunoPrecipitation Assay (RIPA) buffer (50 mM Tris, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na3 VO4 , 1 mM NaF, 1000 mOsm.) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). .. To this cell suspension, 1.5 g glass beads (Sigma-Aldrich, G 9268, 425–600 µm, U.S. sieve) were added, and the mixture was homogenized thrice in a TissueLyser LT (Invitrogen) containing liquid nitrogen for 5 min.

    Article Title: Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
    Article Snippet: The endonuclease activity inhibition by lucanthone was analyzed using standard Lineweaver-Burke Plot to determine if the inhibition was competitive or non-competitive. .. Cleavage of APE1 by lucanthone and CRT0044876 Western blotting was carried out by 7.5% SDS-PAGE of cell extracts (20 µg total protein per lane) either from APE1 overexpresser clone 5 pretreated with 2.5–200 µM concentration of lucanthone/CRT0044876 or recombinant APE1 for 2 h at 37°C in presence of protease inhibitor cocktail (2 tablets (Roche, # 11836153001) containing mixture of several protease inhibitors with broad inhibitory specificity for serine, cysteine and metalloproteases in all systems, dissolved in 20 ml of APE1 buffer). ..

    Article Title: Molecular mode of action of an Acyl Protein thioesterase
    Article Snippet: 35 S-cystein-methionin radiolabeling experiments HeLa cells were transfected with different Myc-APT2 constructs for 24 h, the cells were starved in DMEM HG devoid of Cys/Met for 30 min at 37°C, pulsed with the same medium supplemented with 140 μCi of 35 S Cys/Met (American Radiolabeled Chemicals, Inc.) for 20 min, washed, and incubated in DMEM complete medium for the indicated time of chase before immunoprecipitation as for 3H-palmitic acid radiolabeling experiments. .. Membrane-cytosol separationTo prepare the post-nuclear supernatant (PNS), HeLa cells were harvested, washed with PBS, and homogenized by passage through a 22 G injection needle in HB (2.9 mM imidazole and 250 mM sucrose, pH 7.4) containing a mini tablet protease inhibitor mixture (Roche). .. After centrifugation, the supernatant was collected as HeLa PNS.

    Centrifugation:

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Incubation:

    Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1
    Article Snippet: Putative targets related to apoptosis and closely correlated with mTOR were further subjected to the luciferase verification system. .. PP2A activity assay In brief, cells were suspended in lysis buffer (50-mM Tris-HCl, pH 7.4, 7.5% glycerol, 1-mM EDTA, 150-mM NaCl, 0.5% NP-40, 1-mM Na3 VO4 , and cOmplete Protease Inhibitor [Roche]), cleared of debris via centrifugation for 15 min at 12,000 rpm, and incubated with PP2A or c-Myc antibody, followed by protein A agarose beads provided in the kit. .. After washing, the beads were resuspended in a mixture of PP2A phosphatase reaction buffer and threonine phosphopeptide and incubated for 10 min at 30°C in a shaking incubator, followed by detection of released phosphate with Malachite green solution.

    Binding Assay:

    Article Title: Characterization of Chromoshadow Domain-mediated Binding of Heterochromatin Protein 1? (HP1?) to Histone H3 *
    Article Snippet: 15 N-Labeled CSDα(109–185) was purified on Mono Q and then Superdex 75 gel filtration columns (GE Healthcare). .. GST Pulldown Assays Approximately 5 μg of protein was mixed with a GST fusion protein immobilized on glutathione-Sepharose beads in 250 μl of binding buffer (150 mm NaCl, 20 mm Tris-HCl (pH 7.5), 5 mm DTT, 0.25% Nonidet P-40, and 1× complete protease inhibitor mixture (Roche Applied Science)). .. After incubation at 4 °C for 2 h, the beads were washed four times with 0.7 ml of binding buffer.

    Western Blot:

    Article Title: The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding
    Article Snippet: .. Western blot analysis For western blot analyses, pellets of infected FHK cells or RK/gH1 were resuspended in radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Roche, Mannheim, Germany). .. Sample buffer (1M Tris/HCl, pH 6.8; 0.8% sodium dodecyl sulfate (SDS); 0.4% glycerol; 0.15% β-mercaptoethanol; 0.004% bromophenol blue) was added, the mixture was heated at 95°C for 5 min and proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) exactly as previously described [ ].

    Article Title: Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
    Article Snippet: The endonuclease activity inhibition by lucanthone was analyzed using standard Lineweaver-Burke Plot to determine if the inhibition was competitive or non-competitive. .. Cleavage of APE1 by lucanthone and CRT0044876 Western blotting was carried out by 7.5% SDS-PAGE of cell extracts (20 µg total protein per lane) either from APE1 overexpresser clone 5 pretreated with 2.5–200 µM concentration of lucanthone/CRT0044876 or recombinant APE1 for 2 h at 37°C in presence of protease inhibitor cocktail (2 tablets (Roche, # 11836153001) containing mixture of several protease inhibitors with broad inhibitory specificity for serine, cysteine and metalloproteases in all systems, dissolved in 20 ml of APE1 buffer). ..

    Infection:

    Article Title: The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding
    Article Snippet: .. Western blot analysis For western blot analyses, pellets of infected FHK cells or RK/gH1 were resuspended in radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Roche, Mannheim, Germany). .. Sample buffer (1M Tris/HCl, pH 6.8; 0.8% sodium dodecyl sulfate (SDS); 0.4% glycerol; 0.15% β-mercaptoethanol; 0.004% bromophenol blue) was added, the mixture was heated at 95°C for 5 min and proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) exactly as previously described [ ].

    Radio Immunoprecipitation:

    Article Title: The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding
    Article Snippet: .. Western blot analysis For western blot analyses, pellets of infected FHK cells or RK/gH1 were resuspended in radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Roche, Mannheim, Germany). .. Sample buffer (1M Tris/HCl, pH 6.8; 0.8% sodium dodecyl sulfate (SDS); 0.4% glycerol; 0.15% β-mercaptoethanol; 0.004% bromophenol blue) was added, the mixture was heated at 95°C for 5 min and proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) exactly as previously described [ ].

    Article Title: Membrane Labeling of Coral Gastrodermal Cells by Biotinylation: The Proteomic Identification of Surface Proteins Involving Cnidaria-Dinoflagellate Endosymbiosis
    Article Snippet: After complete drying, grids were observed with a JEM-1400 transmission electron microscope (JEOL, Japan). .. 2D SDS-PAGE analysis of biotinylated proteins Biotinylated SGCs were prepared as described above and suspended in 550 µL modified isotonic RadioImmunoPrecipitation Assay (RIPA) buffer (50 mM Tris, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na3 VO4 , 1 mM NaF, 1000 mOsm.) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). .. To this cell suspension, 1.5 g glass beads (Sigma-Aldrich, G 9268, 425–600 µm, U.S. sieve) were added, and the mixture was homogenized thrice in a TissueLyser LT (Invitrogen) containing liquid nitrogen for 5 min.

    SDS Page:

    Article Title: Membrane Labeling of Coral Gastrodermal Cells by Biotinylation: The Proteomic Identification of Surface Proteins Involving Cnidaria-Dinoflagellate Endosymbiosis
    Article Snippet: After complete drying, grids were observed with a JEM-1400 transmission electron microscope (JEOL, Japan). .. 2D SDS-PAGE analysis of biotinylated proteins Biotinylated SGCs were prepared as described above and suspended in 550 µL modified isotonic RadioImmunoPrecipitation Assay (RIPA) buffer (50 mM Tris, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na3 VO4 , 1 mM NaF, 1000 mOsm.) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). .. To this cell suspension, 1.5 g glass beads (Sigma-Aldrich, G 9268, 425–600 µm, U.S. sieve) were added, and the mixture was homogenized thrice in a TissueLyser LT (Invitrogen) containing liquid nitrogen for 5 min.

    Article Title: Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
    Article Snippet: The endonuclease activity inhibition by lucanthone was analyzed using standard Lineweaver-Burke Plot to determine if the inhibition was competitive or non-competitive. .. Cleavage of APE1 by lucanthone and CRT0044876 Western blotting was carried out by 7.5% SDS-PAGE of cell extracts (20 µg total protein per lane) either from APE1 overexpresser clone 5 pretreated with 2.5–200 µM concentration of lucanthone/CRT0044876 or recombinant APE1 for 2 h at 37°C in presence of protease inhibitor cocktail (2 tablets (Roche, # 11836153001) containing mixture of several protease inhibitors with broad inhibitory specificity for serine, cysteine and metalloproteases in all systems, dissolved in 20 ml of APE1 buffer). ..

    Modification:

    Article Title: Membrane Labeling of Coral Gastrodermal Cells by Biotinylation: The Proteomic Identification of Surface Proteins Involving Cnidaria-Dinoflagellate Endosymbiosis
    Article Snippet: After complete drying, grids were observed with a JEM-1400 transmission electron microscope (JEOL, Japan). .. 2D SDS-PAGE analysis of biotinylated proteins Biotinylated SGCs were prepared as described above and suspended in 550 µL modified isotonic RadioImmunoPrecipitation Assay (RIPA) buffer (50 mM Tris, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na3 VO4 , 1 mM NaF, 1000 mOsm.) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). .. To this cell suspension, 1.5 g glass beads (Sigma-Aldrich, G 9268, 425–600 µm, U.S. sieve) were added, and the mixture was homogenized thrice in a TissueLyser LT (Invitrogen) containing liquid nitrogen for 5 min.

    Concentration Assay:

    Article Title: Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
    Article Snippet: The endonuclease activity inhibition by lucanthone was analyzed using standard Lineweaver-Burke Plot to determine if the inhibition was competitive or non-competitive. .. Cleavage of APE1 by lucanthone and CRT0044876 Western blotting was carried out by 7.5% SDS-PAGE of cell extracts (20 µg total protein per lane) either from APE1 overexpresser clone 5 pretreated with 2.5–200 µM concentration of lucanthone/CRT0044876 or recombinant APE1 for 2 h at 37°C in presence of protease inhibitor cocktail (2 tablets (Roche, # 11836153001) containing mixture of several protease inhibitors with broad inhibitory specificity for serine, cysteine and metalloproteases in all systems, dissolved in 20 ml of APE1 buffer). ..

    Recombinant:

    Article Title: Lucanthone and Its Derivative Hycanthone Inhibit Apurinic Endonuclease-1 (APE1) by Direct Protein Binding
    Article Snippet: The endonuclease activity inhibition by lucanthone was analyzed using standard Lineweaver-Burke Plot to determine if the inhibition was competitive or non-competitive. .. Cleavage of APE1 by lucanthone and CRT0044876 Western blotting was carried out by 7.5% SDS-PAGE of cell extracts (20 µg total protein per lane) either from APE1 overexpresser clone 5 pretreated with 2.5–200 µM concentration of lucanthone/CRT0044876 or recombinant APE1 for 2 h at 37°C in presence of protease inhibitor cocktail (2 tablets (Roche, # 11836153001) containing mixture of several protease inhibitors with broad inhibitory specificity for serine, cysteine and metalloproteases in all systems, dissolved in 20 ml of APE1 buffer). ..

    Injection:

    Article Title: Molecular mode of action of an Acyl Protein thioesterase
    Article Snippet: 35 S-cystein-methionin radiolabeling experiments HeLa cells were transfected with different Myc-APT2 constructs for 24 h, the cells were starved in DMEM HG devoid of Cys/Met for 30 min at 37°C, pulsed with the same medium supplemented with 140 μCi of 35 S Cys/Met (American Radiolabeled Chemicals, Inc.) for 20 min, washed, and incubated in DMEM complete medium for the indicated time of chase before immunoprecipitation as for 3H-palmitic acid radiolabeling experiments. .. Membrane-cytosol separationTo prepare the post-nuclear supernatant (PNS), HeLa cells were harvested, washed with PBS, and homogenized by passage through a 22 G injection needle in HB (2.9 mM imidazole and 250 mM sucrose, pH 7.4) containing a mini tablet protease inhibitor mixture (Roche). .. After centrifugation, the supernatant was collected as HeLa PNS.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Roche anti ha antibody conjugated agarose beads
    The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with <t>anti-myc</t> <t>antibody</t> and <t>agarose-conjugated</t> protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against <t>A3DE-HA</t> and Vif-myc.
    Anti Ha Antibody Conjugated Agarose Beads, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ha antibody conjugated agarose beads/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ha antibody conjugated agarose beads - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    86
    Roche complete protease inhibitor mix
    Soluble JAM-A serum levels do not indicate BBB disturbance in multiple sclerosis and ischemic stroke. A Levels of sJAM-A in serum samples from 45 patients with stable relapsing-remitting MS not receiving secondary prophylactic therapy were measured in duplicates by ELISA and compared to those in 14 untreated MS patients during an acute relapse. B Levels of sJAM-A were measured in serum samples from 13 patients with non-small vessel <t>complete</t> ischemic stroke where a first sample was obtained within the first 3 h of symptom onset and a second sample 24 h after clinical onset. Serum samples were not concentrated before ELISA measurement. Dots represent single values, lines median values. Serum levels in stable and inflammatory-active MS patients were statistically compared by the Mann-Whitney U test, serum levels in stroke patients over time by the Wilcoxon matched pairs test. n.s., not significant.
    Complete Protease Inhibitor Mix, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete protease inhibitor mix/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    complete protease inhibitor mix - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    86
    Roche triton x 100
    The sequence identity of the endogenous proprotein convertase-cleavage motif within Pmel17 is not essential for proper early maturation. A , schematic representation of the IR construct.  B , IR is processed by pPCs to give rise to Mα, Mβ, and HMB45-reactive fibrillogenic fragments. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS, 1% β-mercaptoethanol + protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies.  C , IR displays a relatively normal early maturation. Cells from  B  were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography ( left panel ). Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown ( right panel ).  Error bars  reflect the standard deviation from the mean of two independent experiments.  D , newly synthesized IR localizes to the ER and Golgi apparatus. Cells from  B  were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). A higher magnification of the indicated area is shown as an  inset  within each image.  E , IR is expressed at the cell surface. Cells from  B  were surface labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry ( histograms  on the  left ). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram ( right panel ).
    Triton X 100, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 100/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    triton x 100 - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    Image Search Results


    The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.

    Journal: PLoS ONE

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    doi: 10.1371/journal.pone.0003963

    Figure Lengend Snippet: The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.

    Article Snippet: Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis

    The C-CCD of A3F behaves like the full-length A3F. (A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.

    Journal: PLoS ONE

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    doi: 10.1371/journal.pone.0003963

    Figure Lengend Snippet: The C-CCD of A3F behaves like the full-length A3F. (A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.

    Article Snippet: Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20).

    Techniques: Plasmid Preparation, Immunoprecipitation, Transfection, Expressing, Mutagenesis

    The D 14 RMR 17 and Trp79 domains mediate the interaction between HIV-1 Vif and A3C. Vif DR14/15 and W79 showed reduced interaction with A3C when compared to WT Vif. HEK293T cells were cotransfected with A3C and the control vector, HIV-1 Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysate (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3G-HA and Vif-myc.

    Journal: PLoS ONE

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    doi: 10.1371/journal.pone.0003963

    Figure Lengend Snippet: The D 14 RMR 17 and Trp79 domains mediate the interaction between HIV-1 Vif and A3C. Vif DR14/15 and W79 showed reduced interaction with A3C when compared to WT Vif. HEK293T cells were cotransfected with A3C and the control vector, HIV-1 Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysate (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3G-HA and Vif-myc.

    Article Snippet: Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis

    Soluble JAM-A serum levels do not indicate BBB disturbance in multiple sclerosis and ischemic stroke. A Levels of sJAM-A in serum samples from 45 patients with stable relapsing-remitting MS not receiving secondary prophylactic therapy were measured in duplicates by ELISA and compared to those in 14 untreated MS patients during an acute relapse. B Levels of sJAM-A were measured in serum samples from 13 patients with non-small vessel complete ischemic stroke where a first sample was obtained within the first 3 h of symptom onset and a second sample 24 h after clinical onset. Serum samples were not concentrated before ELISA measurement. Dots represent single values, lines median values. Serum levels in stable and inflammatory-active MS patients were statistically compared by the Mann-Whitney U test, serum levels in stroke patients over time by the Wilcoxon matched pairs test. n.s., not significant.

    Journal: PLoS ONE

    Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown

    doi: 10.1371/journal.pone.0013568

    Figure Lengend Snippet: Soluble JAM-A serum levels do not indicate BBB disturbance in multiple sclerosis and ischemic stroke. A Levels of sJAM-A in serum samples from 45 patients with stable relapsing-remitting MS not receiving secondary prophylactic therapy were measured in duplicates by ELISA and compared to those in 14 untreated MS patients during an acute relapse. B Levels of sJAM-A were measured in serum samples from 13 patients with non-small vessel complete ischemic stroke where a first sample was obtained within the first 3 h of symptom onset and a second sample 24 h after clinical onset. Serum samples were not concentrated before ELISA measurement. Dots represent single values, lines median values. Serum levels in stable and inflammatory-active MS patients were statistically compared by the Mann-Whitney U test, serum levels in stroke patients over time by the Wilcoxon matched pairs test. n.s., not significant.

    Article Snippet: Western Blotting For generation of whole cell protein extracts, cells grown to subconfluency in 25 cm2 flasks were stimulated as indicated, washed with icecold PBS and scraped into radioimmunoprecipitation assay (RIPA) buffer composed of 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% Nonidet P40; 0.25% sodium deoxycholate and 1× Roche COMPLETE® protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany).

    Techniques: Mass Spectrometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    The sequence identity of the endogenous proprotein convertase-cleavage motif within Pmel17 is not essential for proper early maturation. A , schematic representation of the IR construct.  B , IR is processed by pPCs to give rise to Mα, Mβ, and HMB45-reactive fibrillogenic fragments. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS, 1% β-mercaptoethanol + protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies.  C , IR displays a relatively normal early maturation. Cells from  B  were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography ( left panel ). Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown ( right panel ).  Error bars  reflect the standard deviation from the mean of two independent experiments.  D , newly synthesized IR localizes to the ER and Golgi apparatus. Cells from  B  were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). A higher magnification of the indicated area is shown as an  inset  within each image.  E , IR is expressed at the cell surface. Cells from  B  were surface labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry ( histograms  on the  left ). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram ( right panel ).

    Journal: The Journal of Biological Chemistry

    Article Title: Proprotein Convertases Process Pmel17 during Secretion *

    doi: 10.1074/jbc.M110.168088

    Figure Lengend Snippet: The sequence identity of the endogenous proprotein convertase-cleavage motif within Pmel17 is not essential for proper early maturation. A , schematic representation of the IR construct. B , IR is processed by pPCs to give rise to Mα, Mβ, and HMB45-reactive fibrillogenic fragments. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS, 1% β-mercaptoethanol + protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies. C , IR displays a relatively normal early maturation. Cells from B were pulse-labeled for 30 min with 35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography ( left panel ). Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown ( right panel ). Error bars reflect the standard deviation from the mean of two independent experiments. D , newly synthesized IR localizes to the ER and Golgi apparatus. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). A higher magnification of the indicated area is shown as an inset within each image. E , IR is expressed at the cell surface. Cells from B were surface labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry ( histograms on the left ). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram ( right panel ).

    Article Snippet: If the pre-clear had not been performed overnight, the frozen cell pellets were thawed, lysed in 2% Triton X-100 (containing protease inhibitor mixture (Roche)) at 107 cells/ml, and precleared using protein A-Sepharose beads.

    Techniques: Sequencing, Construct, Derivative Assay, Western Blot, Labeling, Immunoprecipitation, Autoradiography, Pulse Chase, Standard Deviation, Synthesized, Immunofluorescence, Flow Cytometry, Cytometry

    Brefeldin A, but not monensin treatment abrogates proprotein convertase-mediated processing of Pmel17. A , monensin treatment does not abrogate proprotein convertase-mediated processing of Pmel17. Mel220 transfectants stably expressing wt-Pmel17 or IR were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. During both labeling and chase, 10 μ m  monensin was included ( sixth  to  ninth  and  14th  to  17th lanes 6–9 ) or no inhibitor was included at all ( first  to  fifth lanes  and  10th  to  13th ). 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography.  B , quantitative PhosphorImager analysis of the pulse-chase data in  A  and a second independent experiment ( supplemental Fig. S5 ) is shown. The figure displays the ratio of the P2 form  versus  the Mα fragment at the indicated time points of chase.  Error bars  reflect the mean ± S.D. of the two independent experiments.  C , quantitative PhosphorImager analysis of the pulse-chase data in  A  and a second independent experiment ( supplemental Fig. S5 ) with maximal levels for each band set to 100% is shown.  Error bars  reflect the mean ± S.D. of two independent experiments.  D , Mel220 transfectants stably expressing wt-Pmel17 were treated or not with 10 μg/ml of brefeldin A ( BFA ) overnight and subsequently analyzed by Western blot using the Pmel17-specific antibody Pep13h.

    Journal: The Journal of Biological Chemistry

    Article Title: Proprotein Convertases Process Pmel17 during Secretion *

    doi: 10.1074/jbc.M110.168088

    Figure Lengend Snippet: Brefeldin A, but not monensin treatment abrogates proprotein convertase-mediated processing of Pmel17. A , monensin treatment does not abrogate proprotein convertase-mediated processing of Pmel17. Mel220 transfectants stably expressing wt-Pmel17 or IR were pulse-labeled for 30 min with 35 S and subsequently chased for the indicated times. During both labeling and chase, 10 μ m monensin was included ( sixth to ninth and 14th to 17th lanes 6–9 ) or no inhibitor was included at all ( first to fifth lanes and 10th to 13th ). 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography. B , quantitative PhosphorImager analysis of the pulse-chase data in A and a second independent experiment ( supplemental Fig. S5 ) is shown. The figure displays the ratio of the P2 form versus the Mα fragment at the indicated time points of chase. Error bars reflect the mean ± S.D. of the two independent experiments. C , quantitative PhosphorImager analysis of the pulse-chase data in A and a second independent experiment ( supplemental Fig. S5 ) with maximal levels for each band set to 100% is shown. Error bars reflect the mean ± S.D. of two independent experiments. D , Mel220 transfectants stably expressing wt-Pmel17 were treated or not with 10 μg/ml of brefeldin A ( BFA ) overnight and subsequently analyzed by Western blot using the Pmel17-specific antibody Pep13h.

    Article Snippet: If the pre-clear had not been performed overnight, the frozen cell pellets were thawed, lysed in 2% Triton X-100 (containing protease inhibitor mixture (Roche)) at 107 cells/ml, and precleared using protein A-Sepharose beads.

    Techniques: Stable Transfection, Expressing, Labeling, Immunoprecipitation, Autoradiography, Pulse Chase, Western Blot

    Surface Pmel17 is already in a proprotein convertase-cleaved state. A , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were incubated on ice with Pmel17-specific antibody HMB50, then extensively washed and lysed in 2% Triton X-100 before protein A-Sepharose beads were added to specifically immunoprecipitate the surface population of Pmel17. Immunoprecipitates ( third, fourth, seventh , and  eighth lanes ) or corresponding total cell lysates ( first, second, fifth , and  sixth lanes ) were analyzed by Western blot using antibody Pep13h. A shorter ( first  to  fourth lanes ) and a longer ( fifth  to  eighth lanes ) exposure of the same membrane is shown. The  dashed line  indicates a position where irrelevant lanes have been removed from the image. The  asterisks  indicate nonspecifically precipitated proteins. Note that the ER P1 form (present in total cell lysate, but not in the surface-IP sample) serves as an internal control in the experiment.  B , newly synthesized Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were pulse-labeled for 30 min with  35 S and subsequently chased for 1 h. Antibody HMB50 was added to the intact cells on ice, before extensive washing, lysis in 2% Triton X-100, and addition of protein A-Sepharose beads to specifically immunoprecipitate the surface population of Pmel17 ( second  and  third lanes ). In parallel, a 2% Triton X-100 total cell lysate ( lane 1 ) was immunoprecipitated with Pmel17-specific antibody HMB50 as described in the legend to   Fig. 1 C . Immunoprecipitates were subsequently analyzed by autoradiography ( upper panel ). For quantification, the ratio of surface-immunoprecipitated ( lane 3 )  versus  total cell-associated Pmel17 forms (P1, P2, Mα, and Mβ) ( first lane ) was determined. Note that this percentage is almost equally low for the P2 form and the ER-located P1 form, which serves as an indicator for background precipitation ( gray shaded area ). Only Mα and Mβ are precipitated above this background.  Error bars  reflect the mean ± S.D. of two independe nt experiments. The  dashed line  indicates a position where irrelevant lanes have been removed from the image.  C , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Cells from  A  were surface-biotinylated at room temperature according to the protocol of the manufacturer ( left panel ) or at 4 °C to avoid any residual endocytosis during this step ( right panel ). Subsequently, biotinylated surface proteins were precipitated using avidin-agarose. Protein G-agarose was used as a specificity control. These samples ( third  and  fourth lanes  in  left panel  and  third to fifth  in  right panel ) or total cell lysates ( first  and  second lanes  in  left  and  right panels ) were analyzed by Western blot using Pmel17-specific antibodies Pep13h and Pmel-N. The  horizontal dashed lines  separate the regions of the membrane that were incubated with antibody Pmel-N ( upper part ) and antibody Pep13h ( lower part ), respectively. The  vertical dotted lines  separate longer ( right part ) or shorter ( left part ) exposures of the same membrane. Note that the ER P1 form (present in total cell lysate, but only marginally in the avidin-precipitation samples) serves as an internal control in the experiment.

    Journal: The Journal of Biological Chemistry

    Article Title: Proprotein Convertases Process Pmel17 during Secretion *

    doi: 10.1074/jbc.M110.168088

    Figure Lengend Snippet: Surface Pmel17 is already in a proprotein convertase-cleaved state. A , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were incubated on ice with Pmel17-specific antibody HMB50, then extensively washed and lysed in 2% Triton X-100 before protein A-Sepharose beads were added to specifically immunoprecipitate the surface population of Pmel17. Immunoprecipitates ( third, fourth, seventh , and eighth lanes ) or corresponding total cell lysates ( first, second, fifth , and sixth lanes ) were analyzed by Western blot using antibody Pep13h. A shorter ( first to fourth lanes ) and a longer ( fifth to eighth lanes ) exposure of the same membrane is shown. The dashed line indicates a position where irrelevant lanes have been removed from the image. The asterisks indicate nonspecifically precipitated proteins. Note that the ER P1 form (present in total cell lysate, but not in the surface-IP sample) serves as an internal control in the experiment. B , newly synthesized Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Mel220 transfectants stably expressing wt-Pmel17 were pulse-labeled for 30 min with 35 S and subsequently chased for 1 h. Antibody HMB50 was added to the intact cells on ice, before extensive washing, lysis in 2% Triton X-100, and addition of protein A-Sepharose beads to specifically immunoprecipitate the surface population of Pmel17 ( second and third lanes ). In parallel, a 2% Triton X-100 total cell lysate ( lane 1 ) was immunoprecipitated with Pmel17-specific antibody HMB50 as described in the legend to Fig. 1 C . Immunoprecipitates were subsequently analyzed by autoradiography ( upper panel ). For quantification, the ratio of surface-immunoprecipitated ( lane 3 ) versus total cell-associated Pmel17 forms (P1, P2, Mα, and Mβ) ( first lane ) was determined. Note that this percentage is almost equally low for the P2 form and the ER-located P1 form, which serves as an indicator for background precipitation ( gray shaded area ). Only Mα and Mβ are precipitated above this background. Error bars reflect the mean ± S.D. of two independe nt experiments. The dashed line indicates a position where irrelevant lanes have been removed from the image. C , all Pmel17 at the cell surface is already in a proprotein convertase-cleaved state. Cells from A were surface-biotinylated at room temperature according to the protocol of the manufacturer ( left panel ) or at 4 °C to avoid any residual endocytosis during this step ( right panel ). Subsequently, biotinylated surface proteins were precipitated using avidin-agarose. Protein G-agarose was used as a specificity control. These samples ( third and fourth lanes in left panel and third to fifth in right panel ) or total cell lysates ( first and second lanes in left and right panels ) were analyzed by Western blot using Pmel17-specific antibodies Pep13h and Pmel-N. The horizontal dashed lines separate the regions of the membrane that were incubated with antibody Pmel-N ( upper part ) and antibody Pep13h ( lower part ), respectively. The vertical dotted lines separate longer ( right part ) or shorter ( left part ) exposures of the same membrane. Note that the ER P1 form (present in total cell lysate, but only marginally in the avidin-precipitation samples) serves as an internal control in the experiment.

    Article Snippet: If the pre-clear had not been performed overnight, the frozen cell pellets were thawed, lysed in 2% Triton X-100 (containing protease inhibitor mixture (Roche)) at 107 cells/ml, and precleared using protein A-Sepharose beads.

    Techniques: Stable Transfection, Expressing, Incubation, Western Blot, Synthesized, Labeling, Lysis, Immunoprecipitation, Autoradiography, Avidin-Biotin Assay

    A soluble Pmel17 mutant gets secreted from cells in a proprotein convertase-cleaved form. A , schematic representation of the sPmel17-myc construct.  B , only the ER-associated P1 form can be detected for sPmel17-myc inside the cells at steady-state. Membrane lysates of Mel220 transfectants stably expressing sPmel17-myc were prepared as in   Fig. 1 B  and analyzed by Western blot using Pmel17-specific antibodies, myc-specific antibodies, or tapasin-specific antibodies for control.  C , almost all intracellular sPmel17-myc is localized to the ER. Cells from  B  were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 ( Pmel-N ), mature Pmel17 ( HMB50 ), or the myc-tag ( 9E10 ).  D , soluble sPmel17-myc gets secreted into the culture medium. Mel220 transfectants stably expressing sPmel17-myc were pulse-labeled for 30 min with  35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates ( left panel ) or culture supernatants ( right panel ) were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography. The  dashed lines  indicate positions where irrelevant lanes have been removed from the image. The  pound symbol  indicates background levels of the precipitated P1 form. The  asterisks  indicate nonspecifically precipitated proteins.  E , quantitative PhosphorImager analysis of the pulse-chase data in  D  and a second independent experiment with maximal levels for each band set to 100%.  Error bars  reflect the mean ± S.D. of these two independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Proprotein Convertases Process Pmel17 during Secretion *

    doi: 10.1074/jbc.M110.168088

    Figure Lengend Snippet: A soluble Pmel17 mutant gets secreted from cells in a proprotein convertase-cleaved form. A , schematic representation of the sPmel17-myc construct. B , only the ER-associated P1 form can be detected for sPmel17-myc inside the cells at steady-state. Membrane lysates of Mel220 transfectants stably expressing sPmel17-myc were prepared as in Fig. 1 B and analyzed by Western blot using Pmel17-specific antibodies, myc-specific antibodies, or tapasin-specific antibodies for control. C , almost all intracellular sPmel17-myc is localized to the ER. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 ( Pmel-N ), mature Pmel17 ( HMB50 ), or the myc-tag ( 9E10 ). D , soluble sPmel17-myc gets secreted into the culture medium. Mel220 transfectants stably expressing sPmel17-myc were pulse-labeled for 30 min with 35 S and subsequently chased for the indicated times. 2% Triton X-100 lysates ( left panel ) or culture supernatants ( right panel ) were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS by boiling for 5 min, and analyzed by autoradiography. The dashed lines indicate positions where irrelevant lanes have been removed from the image. The pound symbol indicates background levels of the precipitated P1 form. The asterisks indicate nonspecifically precipitated proteins. E , quantitative PhosphorImager analysis of the pulse-chase data in D and a second independent experiment with maximal levels for each band set to 100%. Error bars reflect the mean ± S.D. of these two independent experiments.

    Article Snippet: If the pre-clear had not been performed overnight, the frozen cell pellets were thawed, lysed in 2% Triton X-100 (containing protease inhibitor mixture (Roche)) at 107 cells/ml, and precleared using protein A-Sepharose beads.

    Techniques: Mutagenesis, Construct, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Synthesized, Labeling, Immunoprecipitation, Autoradiography, Pulse Chase