protease inhibitor mixture  (Roche)


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    Structured Review

    Roche protease inhibitor mixture
    Protease Inhibitor Mixture, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, 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 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mixture - by Bioz Stars, 2021-05
    96/100 stars

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    Protease Inhibitor:

    Article Title: Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration *
    Article Snippet: PYK2-SUMO1 Site Mapping 4E107 HeLa cells were cotransfected with V5-PYK2, MYC-SUMO1, and FLAG-PIAS1 using Fugene 6 transfection reagent (Promega). .. After 48 h, cells were rinsed with warm PBS then lysed with RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 1% Triton-X-100) containing 0.5 mm PMSF, Roche Protease Inhibitor Mixture, 20 mm N -ethylmaleimide and 1% SDS. .. The concentrated lysate was spun down to clear debris and the supernatant was diluted with RIPA without SDS, to a final concentration of 0.2% SDS.

    Article Title: Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c
    Article Snippet: Nuclei isolation and micrococcal nuclease (MNase; Roche; 10107921001) digestion were performed following a previously published protocol ( ). .. After MNase digestion, the nuclei were lysed with a lysis buffer (50 mM Tris⋅HCl pH 7.5, 137 mM NaCl, 0.5% Triton X-100, 0.5% CHAPS, 10% (vol/vol) glycerol, and Roche Protease Inhibitor mixture). .. For HA-tagged proteins, the nuclear lysates were incubated with agarose conjugated with an anti-HA antibody (Sigma-Aldrich; A2095).

    Article Title: Role of lysine methylation of NF-?B in differential gene regulation
    Article Snippet: The fresh pellet (108 cells) was then resuspended on ice in 5 mL of Farnham Lysis Buffer, and the crude nuclear preparation was collected by centrifugation at 4 °C. .. The pellets were then resuspended into 5 mL of RIPA buffer [1× PBS, 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, plus Roche Protease Inhibitor Mixture] and 1 mL of aliquots were transferred to 1.5 mL Eppendorf tubes (2 × 107 cells/mL equivalent). .. Each 1.0 mL CHIP sample was sonicated on ice at Power output 5 W, four times for 30 s each, with at least 30 s of cooling on ice between each sonication, to obtain an average length of the sheared chromatin of about 250 bp or less, with a range of about 100–600 bp.

    Article Title: Role of lysine methylation of NF-?B in differential gene regulation
    Article Snippet: The cross-linking reaction was then stopped by adding glycine to a final concentration of 0.125 M. Media was removed from plates and cells were washed with an equal volume of cold 1 × PBS (PBS, Thermo Scientific). .. The buffer was then removed by aspiration and 5–8 mL of cold Farnham lysis buffer [5 mM Pipes, pH 8.0, 85 mM KCl, 0.5% (vol/vol) Nonidet P-40, plus Roche Protease Inhibitor Mixture] was then added to the dish. ..

    Article Title: Somatic sex determination in Caenorhabditis elegans is modulated by SUP-26 repression of tra-2 translation
    Article Snippet: Fluorescence images were subjected to deconvolution analysis using the Slidebook 5.0 software program (Intelligent Imaging Innovations). .. Mixed-stage animals were harvested from nematode growth media (NGM) agar plates and lysed by sonication (3 × 10 s) in a buffer containing 250 mM NaCl, 100 mM Tris·HCl (pH 7.4), 1 mM EDTA, 5 mM DTT, 0.1% Triton X-100, 1% PMSF, and Roche Complete Protease Inhibitor Mixture. .. GST-SUP-26 fusion proteins were expressed and purified as described above.

    Article Title: Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase
    Article Snippet: Approximately 100 mg of either control or stressed Arabidopsis leaves were frozen in liquid nitrogen and ground to a fine powder with a 1/8′′ steel ball with the TissueLyzer II (Qiagen). .. Native proteins were resuspended in ice-cold 50 mM Tris⋅HCl, pH 7.5, supplemented with 1% PVP 360 and 1× Roche Protease Inhibitor Mixture (Roche). .. Cellular debris was removed by centrifugation at 4 °C, and the proteins in the supernatant were quantified by Bradford assay.

    Article Title: Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration *
    Article Snippet: 4E107 HeLa cells were cotransfected with V5-PYK2, MYC-SUMO1, and FLAG-PIAS1 using Fugene 6 transfection reagent (Promega). .. After 48 h, cells were rinsed with warm PBS then lysed with RIPA buffer (50 m m Tris-HCl, pH 7.4, 150 m m NaCl, 2 m m EDTA, 1% Triton-X-100) containing 0.5 m m PMSF, Roche Protease Inhibitor Mixture, 20 m m N -ethylmaleimide and 1% SDS. .. The concentrated lysate was spun down to clear debris and the supernatant was diluted with RIPA without SDS, to a final concentration of 0.2% SDS.

    Article Title: Distribution of SET/I2PP2A protein in gastrointestinal tissues
    Article Snippet: .. Tissue samples were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 5 mM EGTA, 1% Triton X100, 1 mM Na3 VO4 , 20 mM sodium pyrophosphate, and Roche Complete protease inhibitor mixture. .. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Fujifilm, Osaka, Japan).

    Lysis:

    Article Title: Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c
    Article Snippet: Nuclei isolation and micrococcal nuclease (MNase; Roche; 10107921001) digestion were performed following a previously published protocol ( ). .. After MNase digestion, the nuclei were lysed with a lysis buffer (50 mM Tris⋅HCl pH 7.5, 137 mM NaCl, 0.5% Triton X-100, 0.5% CHAPS, 10% (vol/vol) glycerol, and Roche Protease Inhibitor mixture). .. For HA-tagged proteins, the nuclear lysates were incubated with agarose conjugated with an anti-HA antibody (Sigma-Aldrich; A2095).

    Article Title: Role of lysine methylation of NF-?B in differential gene regulation
    Article Snippet: The cross-linking reaction was then stopped by adding glycine to a final concentration of 0.125 M. Media was removed from plates and cells were washed with an equal volume of cold 1 × PBS (PBS, Thermo Scientific). .. The buffer was then removed by aspiration and 5–8 mL of cold Farnham lysis buffer [5 mM Pipes, pH 8.0, 85 mM KCl, 0.5% (vol/vol) Nonidet P-40, plus Roche Protease Inhibitor Mixture] was then added to the dish. ..

    Sonication:

    Article Title: Somatic sex determination in Caenorhabditis elegans is modulated by SUP-26 repression of tra-2 translation
    Article Snippet: Fluorescence images were subjected to deconvolution analysis using the Slidebook 5.0 software program (Intelligent Imaging Innovations). .. Mixed-stage animals were harvested from nematode growth media (NGM) agar plates and lysed by sonication (3 × 10 s) in a buffer containing 250 mM NaCl, 100 mM Tris·HCl (pH 7.4), 1 mM EDTA, 5 mM DTT, 0.1% Triton X-100, 1% PMSF, and Roche Complete Protease Inhibitor Mixture. .. GST-SUP-26 fusion proteins were expressed and purified as described above.

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    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
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    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, 2021-05
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    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
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    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