protease inhibitors  (Roche)


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    Structured Review

    Roche protease inhibitors
    Protease Inhibitors, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 7493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protease inhibitors/product/Roche
    Average 92 stars, based on 7493 article reviews
    Price from $9.99 to $1999.99
    protease inhibitors - by Bioz Stars, 2020-09
    92/100 stars

    Images

    Related Articles

    Radio Immunoprecipitation:

    Article Title: Microenvironment-Derived FGF-2 Stimulates Renal Cell Carcinoma Cell Proliferation through Modulation of p27Kip1: Implications for Spatial Niche Formation and Functional Intratumoral Heterogeneity
    Article Snippet: .. Immunoblot Analysis Cells were lysed in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) containing protease inhibitors and phosphatase inhibitors (Roche). .. A total of 30 μg of protein was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane.

    Homogenization:

    Article Title: Oleic acid-based nanosystems for mitigating acute respiratory distress syndrome in mice through neutrophil suppression: how the particulate size affects therapeutic efficiency
    Article Snippet: .. The tissue was extracted with buffer containing complete protease inhibitors under homogenization (MagNA Lyser, Roche). .. The supernatant was taken to measure TNF-α, IL-1β, IL-6, and CXCL-2 employing commercial kits (BioLegend).

    Protease Inhibitor:

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
    Article Snippet: .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare) .. KinTek quench-flow apparatus (model RQF-3, KinTek Corporation) (requirement depends on transcription factor dynamics; see Section 4), exit tubing of various lengths (see for examples)

    Blocking Assay:

    Article Title: Identification of Sugar Residues Involved in the Binding of TGEV to Porcine Brush Border Membranes
    Article Snippet: .. Protease inhibitors: leupeptin (Roche), Pefa Block® SC (Roche). ..

    Incubation:

    Article Title: Time-dependent Pax3-mediated chromatin remodeling and cooperation with Six4 and Tead2 specify the skeletal myogenic lineage in developing mesoderm
    Article Snippet: .. Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors (50 mM HEPES KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 + complete-mini [Roche]) for 10 minutes at +4°C followed by incubation in buffer LB2 supplemented with protease inhibitors (10 mM Tris-HCl [pH 8], 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA + complete-mini [Roche]) for 10 minutes at +4°C. .. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors (10 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine + complete-mini [Roche]) and then sonicated.

    other:

    Article Title: Lipid Raft-Redox Signaling Platforms in Plasma Membrane
    Article Snippet: One tablet “complete” protease inhibitors dissolved in 1-ml dH2 O (Roche, Branchburg, NJ) MBS buffer containing 1-mM Na3 VO4 , 1-mM phenylmethylsulfonyl fluoride, and “complete” protease inhibitors (1:50 dilution) (solution A).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
    Article Snippet: .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare) .. KinTek quench-flow apparatus (model RQF-3, KinTek Corporation) (requirement depends on transcription factor dynamics; see Section 4), exit tubing of various lengths (see for examples)

    Western Blot:

    Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
    Article Snippet: .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare) .. KinTek quench-flow apparatus (model RQF-3, KinTek Corporation) (requirement depends on transcription factor dynamics; see Section 4), exit tubing of various lengths (see for examples)

    Article Title: Ciprofloxacin promotes polarization of CD86+CD206− macrophages to suppress liver cancer
    Article Snippet: .. Western blottingFor the western blot analyses, RIPA buffer containing protease inhibitors and phosphatase inhibitors (Roche Diagnostics) was used to prepare whole-cell lysates. .. Protein (20 µg) from the lysates was separated by 10% SDS-PAGE and transferred to a PVDF membrane (0.45 µm, EMD Millipore).

    Sonication:

    Article Title: SUMOylation Inhibits SF-1 Activity by Reducing CDK7-Mediated Serine 203 Phosphorylation ▿
    Article Snippet: .. After 48 h, cells were harvested in 700 μl lysis buffer (500 mM NaCl, 10 mM imidazole, 45 mM Na2 HPO4 , 5 mM Na2 H2 PO4 , 8 M urea, pH 8) containing complete protease inhibitors without EDTA (1 tablet/10 ml; Roche) and sonicated. .. The lysates were cleared and incubated with 100 μl of 50% Ni2+ -nitrilotriacetic acid agarose (Qiagen) at room temperature for 60 min on a rotator.

    Lysis:

    Article Title: Time-dependent Pax3-mediated chromatin remodeling and cooperation with Six4 and Tead2 specify the skeletal myogenic lineage in developing mesoderm
    Article Snippet: .. Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors (50 mM HEPES KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 + complete-mini [Roche]) for 10 minutes at +4°C followed by incubation in buffer LB2 supplemented with protease inhibitors (10 mM Tris-HCl [pH 8], 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA + complete-mini [Roche]) for 10 minutes at +4°C. .. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors (10 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine + complete-mini [Roche]) and then sonicated.

    Article Title: Microenvironment-Derived FGF-2 Stimulates Renal Cell Carcinoma Cell Proliferation through Modulation of p27Kip1: Implications for Spatial Niche Formation and Functional Intratumoral Heterogeneity
    Article Snippet: .. Immunoblot Analysis Cells were lysed in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) containing protease inhibitors and phosphatase inhibitors (Roche). .. A total of 30 μg of protein was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane.

    Article Title: SUMOylation Inhibits SF-1 Activity by Reducing CDK7-Mediated Serine 203 Phosphorylation ▿
    Article Snippet: .. After 48 h, cells were harvested in 700 μl lysis buffer (500 mM NaCl, 10 mM imidazole, 45 mM Na2 HPO4 , 5 mM Na2 H2 PO4 , 8 M urea, pH 8) containing complete protease inhibitors without EDTA (1 tablet/10 ml; Roche) and sonicated. .. The lysates were cleared and incubated with 100 μl of 50% Ni2+ -nitrilotriacetic acid agarose (Qiagen) at room temperature for 60 min on a rotator.

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    Roche proteomic analysis
    Differentially regulated mitochondrial proteins identified in patient iNPCs. Comparative <t>proteomic</t> analysis of mitochondrial proteins was performed on three iNPC cells lines (patient 1, patient 2 and control). A total of 85 differentially expressed mitochondrial proteins were identified in comparison between control and patient groups. Screen shot of the STRING (Search Tool for the Retrieval of Interacting Proteins) showed a network of differentially expressed proteins in cells of patient 2 ( A ) and patient 1. ( B ) Black circles indicated protein clusters directly affecting mitochondrial function. Red circles represent protein clusters related to vesicle-associated membrane proteins and its downstream pathways. Blue circle represents fatty acid oxidation pathway. The interactions are shown in confidence view. Thicker lines represent a stronger association. The proteins are identified by their gene names located near each sphere.
    Proteomic Analysis, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche adult spata7 gfp transgenic
    <t>SPATA7</t> extends AHI1 localization apical of the TZ in primary cilia. (a) Endogenous AHI1 (white) localizes at the TZ of the primary cilium, marked by acetylated α-tubulin in hTERT-RPE1 cells (left). Ectopic expression of <t>hSPATA7-GFP</t> (right) induces apical extension of the endogenous AHI1 localization signal beyond the TZ into the ciliary axoneme (subsets display zoomed images of AHI1 [white], acetylated α-tubulin [red], SPATA7-GFP, and DAPI). Bars: (main images) 10 µm; (insets) 2 µm. (b) Model displaying the CC zones PSTZ at the DCC and PCC that are visible in the Spata7 mutant retinae. The CC is comprised of PCC, a region homologous to the TZ of the primary cilium, and PSTZ, a specialized photoreceptor-specific zone of the CC that is crucial for maintenance of the CC microtubule core. OS, outer segment.
    Adult Spata7 Gfp Transgenic, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche dpf larvae
    PIK3C3 deficiency induces polarity defects in IECs. a Immunofluorescence staining of gut cross sections at 8 <t>dpf.</t> In WT embryos, columnar IECs are arranged into a sheet and E-Cadherin are laterally distributed. IECs become disorganized and E-Cadherin is endocytosed and forms intracellular aggregates in the mutants. LAMP1 is a lysosome marker and it does not co-localize with E-Cadherin aggregates. Other polarity-related proteins including Na,K-ATPase alpha 1 subunit (ATP1A1) and tight junction protein ZO-1 also lose their polarized distributions in the mutant guts. Scale bar, 20 µm. b Western blot analysis for the expression levels of E-Cadherin and ZO-1 in the dissected digestive tracts from 8 dpf embryos. Beta actin is the loading control. c qRT-PCR analysis of cdh1 and zo-1 . Assays are performed as described in Fig. 3f . d Knockdown efficiency of PIK3C3 by siRNA in <t>Caco2</t> cells as determined by qRT-PCR. ACTB is the internal control and data represent mean ± SD from three biological repeats (*** p
    Dpf Larvae, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche recombinant bs pbp1 proteins
    The Sp PBP2a minidomain is not α-helical but still interacts with Sp GpsB through conserved arginines. a Arginine residues of Sp PBP2a play a key role in binding to Sp GpsB. Unless otherwise indicated, the fluorescence polarisation binding curves represent the interaction of TAMRA-labelled Sp PBP2a 23-45 peptides with wildtype Sp GpsB 1-63 . The relevant dissociation constants are listed in Supplementary Table 1 . b The structure of the Sp GpsB 4-63 : Sp PBP2a 27-40 complex reveals the critical role of Sp PBP2a arginines for the interaction with Sp GpsB. In this cartoon, Sp GpsB 4-63 is coloured cyan, and the Sp PBP2a 27-40 peptide is coloured yellow (molecule 1) and green (molecule 2). The sidechains of Arg8 and Arg11 from the Bs GpsB 5-64 : Bs <t>PBP1</t> 1-17 complex are shown as red sticks after a global superimposition of equivalent GpsB atoms. In molecule 1, Sp PBP2a Arg31 and Sp PBP2a Arg36 superimpose with Bs GpsB 5-64 Arg8 and Bs GpsB 5-64 Arg11 whereas molecule 2 accommodates Sp PBP2a Arg33 and Sp PBP2a Arg36 . c , d Close-up view of the interactions of Sp PBP2a from molecule 1 ( c ) and 2 ( d ) with Sp GpsB 4-63 . Key interfacial sidechains and backbone atoms are represented in stick format; Sp GpsB 4-63 is coloured cyan and Sp PBP2A 27-40 is coloured green. The van der Waals’ interactions between Sp GpsB Leu32 and Sp PBP1 Arg31 (molecule 1) and Sp PBP1 Arg33 (molecule 2) are in yellow. The carbonyl oxygens of Sp GpsB Ile11 , Sp GpsB Phe12 , Sp GpsB Glu13 and Sp GpsB Gln14 are denoted by respective red numerals
    Recombinant Bs Pbp1 Proteins, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Differentially regulated mitochondrial proteins identified in patient iNPCs. Comparative proteomic analysis of mitochondrial proteins was performed on three iNPC cells lines (patient 1, patient 2 and control). A total of 85 differentially expressed mitochondrial proteins were identified in comparison between control and patient groups. Screen shot of the STRING (Search Tool for the Retrieval of Interacting Proteins) showed a network of differentially expressed proteins in cells of patient 2 ( A ) and patient 1. ( B ) Black circles indicated protein clusters directly affecting mitochondrial function. Red circles represent protein clusters related to vesicle-associated membrane proteins and its downstream pathways. Blue circle represents fatty acid oxidation pathway. The interactions are shown in confidence view. Thicker lines represent a stronger association. The proteins are identified by their gene names located near each sphere.

    Journal: Human Molecular Genetics

    Article Title: Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons

    doi: 10.1093/hmg/ddy127

    Figure Lengend Snippet: Differentially regulated mitochondrial proteins identified in patient iNPCs. Comparative proteomic analysis of mitochondrial proteins was performed on three iNPC cells lines (patient 1, patient 2 and control). A total of 85 differentially expressed mitochondrial proteins were identified in comparison between control and patient groups. Screen shot of the STRING (Search Tool for the Retrieval of Interacting Proteins) showed a network of differentially expressed proteins in cells of patient 2 ( A ) and patient 1. ( B ) Black circles indicated protein clusters directly affecting mitochondrial function. Red circles represent protein clusters related to vesicle-associated membrane proteins and its downstream pathways. Blue circle represents fatty acid oxidation pathway. The interactions are shown in confidence view. Thicker lines represent a stronger association. The proteins are identified by their gene names located near each sphere.

    Article Snippet: Proteomic analysis: sample preparation Samples (two biological replicates per condition) were solubilized in 1% SDS (w/v), 50 mM Tris–Cl pH 7.8, 150 mM NaCl, supplemented with protease inhibitor Complete Mini and phosphatase inhibitor cocktail PhosSTOP (Roche, Switzerland).

    Techniques:

    SPATA7 extends AHI1 localization apical of the TZ in primary cilia. (a) Endogenous AHI1 (white) localizes at the TZ of the primary cilium, marked by acetylated α-tubulin in hTERT-RPE1 cells (left). Ectopic expression of hSPATA7-GFP (right) induces apical extension of the endogenous AHI1 localization signal beyond the TZ into the ciliary axoneme (subsets display zoomed images of AHI1 [white], acetylated α-tubulin [red], SPATA7-GFP, and DAPI). Bars: (main images) 10 µm; (insets) 2 µm. (b) Model displaying the CC zones PSTZ at the DCC and PCC that are visible in the Spata7 mutant retinae. The CC is comprised of PCC, a region homologous to the TZ of the primary cilium, and PSTZ, a specialized photoreceptor-specific zone of the CC that is crucial for maintenance of the CC microtubule core. OS, outer segment.

    Journal: The Journal of Cell Biology

    Article Title: SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium

    doi: 10.1083/jcb.201712117

    Figure Lengend Snippet: SPATA7 extends AHI1 localization apical of the TZ in primary cilia. (a) Endogenous AHI1 (white) localizes at the TZ of the primary cilium, marked by acetylated α-tubulin in hTERT-RPE1 cells (left). Ectopic expression of hSPATA7-GFP (right) induces apical extension of the endogenous AHI1 localization signal beyond the TZ into the ciliary axoneme (subsets display zoomed images of AHI1 [white], acetylated α-tubulin [red], SPATA7-GFP, and DAPI). Bars: (main images) 10 µm; (insets) 2 µm. (b) Model displaying the CC zones PSTZ at the DCC and PCC that are visible in the Spata7 mutant retinae. The CC is comprised of PCC, a region homologous to the TZ of the primary cilium, and PSTZ, a specialized photoreceptor-specific zone of the CC that is crucial for maintenance of the CC microtubule core. OS, outer segment.

    Article Snippet: IP and liquid chromatography (LC)–tandem MS (MS/MS) analysis For coimmunoprecipitation experiments, retinae from five adult Spata7 -GFP transgenic and five adult WT mice (per replicate across three replicates) were lysed in the IP buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, and protease inhibitor cocktail tablets [11836170007; Roche]) by homogenization followed by sonication.

    Techniques: Expressing, Droplet Countercurrent Chromatography, Periodic Counter-current Chromatography, Mutagenesis

    SPATA7 interacts with members of the NPHP–RPGR complex. (a) Distribution of mean GFP/WT Skyline peptide fold change of top candidate interacting partners of SPATA7 observed over three repeats. The asterisk signifies candidates that are enriched > 1,000-fold in the GFP IP fraction in comparison with the WT control fraction. (b) The interaction between SPATA7 and representative RPGR was confirmed in vitro by the BiFC assay. HEK293T cells were transfected with either SPATA7-CVN alone (top) or together with RPGR-VC constructs (bottom). Strong fluorescence signals (green) were observed in cells expressing both SPATA7 and RPGR, which are quantified in c. Bar, 20 µm. (c) The percentage of BiFC-positive cells for all candidate-interacting partners that are known TZ module members was quantified by FACS ( t test) in comparison with untransfected cells and SPATA7 alone. Error bars show means ± SEM.

    Journal: The Journal of Cell Biology

    Article Title: SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium

    doi: 10.1083/jcb.201712117

    Figure Lengend Snippet: SPATA7 interacts with members of the NPHP–RPGR complex. (a) Distribution of mean GFP/WT Skyline peptide fold change of top candidate interacting partners of SPATA7 observed over three repeats. The asterisk signifies candidates that are enriched > 1,000-fold in the GFP IP fraction in comparison with the WT control fraction. (b) The interaction between SPATA7 and representative RPGR was confirmed in vitro by the BiFC assay. HEK293T cells were transfected with either SPATA7-CVN alone (top) or together with RPGR-VC constructs (bottom). Strong fluorescence signals (green) were observed in cells expressing both SPATA7 and RPGR, which are quantified in c. Bar, 20 µm. (c) The percentage of BiFC-positive cells for all candidate-interacting partners that are known TZ module members was quantified by FACS ( t test) in comparison with untransfected cells and SPATA7 alone. Error bars show means ± SEM.

    Article Snippet: IP and liquid chromatography (LC)–tandem MS (MS/MS) analysis For coimmunoprecipitation experiments, retinae from five adult Spata7 -GFP transgenic and five adult WT mice (per replicate across three replicates) were lysed in the IP buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, and protease inhibitor cocktail tablets [11836170007; Roche]) by homogenization followed by sonication.

    Techniques: In Vitro, Bimolecular Fluorescence Complementation Assay, Transfection, Construct, Fluorescence, Expressing, FACS

    PIK3C3 deficiency induces polarity defects in IECs. a Immunofluorescence staining of gut cross sections at 8 dpf. In WT embryos, columnar IECs are arranged into a sheet and E-Cadherin are laterally distributed. IECs become disorganized and E-Cadherin is endocytosed and forms intracellular aggregates in the mutants. LAMP1 is a lysosome marker and it does not co-localize with E-Cadherin aggregates. Other polarity-related proteins including Na,K-ATPase alpha 1 subunit (ATP1A1) and tight junction protein ZO-1 also lose their polarized distributions in the mutant guts. Scale bar, 20 µm. b Western blot analysis for the expression levels of E-Cadherin and ZO-1 in the dissected digestive tracts from 8 dpf embryos. Beta actin is the loading control. c qRT-PCR analysis of cdh1 and zo-1 . Assays are performed as described in Fig. 3f . d Knockdown efficiency of PIK3C3 by siRNA in Caco2 cells as determined by qRT-PCR. ACTB is the internal control and data represent mean ± SD from three biological repeats (*** p

    Journal: Nature Communications

    Article Title: Deficiency in class III PI3-kinase confers postnatal lethality with IBD-like features in zebrafish

    doi: 10.1038/s41467-018-05105-8

    Figure Lengend Snippet: PIK3C3 deficiency induces polarity defects in IECs. a Immunofluorescence staining of gut cross sections at 8 dpf. In WT embryos, columnar IECs are arranged into a sheet and E-Cadherin are laterally distributed. IECs become disorganized and E-Cadherin is endocytosed and forms intracellular aggregates in the mutants. LAMP1 is a lysosome marker and it does not co-localize with E-Cadherin aggregates. Other polarity-related proteins including Na,K-ATPase alpha 1 subunit (ATP1A1) and tight junction protein ZO-1 also lose their polarized distributions in the mutant guts. Scale bar, 20 µm. b Western blot analysis for the expression levels of E-Cadherin and ZO-1 in the dissected digestive tracts from 8 dpf embryos. Beta actin is the loading control. c qRT-PCR analysis of cdh1 and zo-1 . Assays are performed as described in Fig. 3f . d Knockdown efficiency of PIK3C3 by siRNA in Caco2 cells as determined by qRT-PCR. ACTB is the internal control and data represent mean ± SD from three biological repeats (*** p

    Article Snippet: Western blot Caco2 cells or micro-dissected digestive tracts of 8 dpf larvae were collected in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS) and SDS sample buffer (63 mM Tris–HCl pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 3.5% SDS, and 0.01% bromophenol blue) containing protease inhibitor cocktail (04693132001, Roche) plus 1 mM PMSF (78830, Sigma).

    Techniques: Immunofluorescence, Staining, Marker, Mutagenesis, Western Blot, Expressing, Quantitative RT-PCR

    The Sp PBP2a minidomain is not α-helical but still interacts with Sp GpsB through conserved arginines. a Arginine residues of Sp PBP2a play a key role in binding to Sp GpsB. Unless otherwise indicated, the fluorescence polarisation binding curves represent the interaction of TAMRA-labelled Sp PBP2a 23-45 peptides with wildtype Sp GpsB 1-63 . The relevant dissociation constants are listed in Supplementary Table 1 . b The structure of the Sp GpsB 4-63 : Sp PBP2a 27-40 complex reveals the critical role of Sp PBP2a arginines for the interaction with Sp GpsB. In this cartoon, Sp GpsB 4-63 is coloured cyan, and the Sp PBP2a 27-40 peptide is coloured yellow (molecule 1) and green (molecule 2). The sidechains of Arg8 and Arg11 from the Bs GpsB 5-64 : Bs PBP1 1-17 complex are shown as red sticks after a global superimposition of equivalent GpsB atoms. In molecule 1, Sp PBP2a Arg31 and Sp PBP2a Arg36 superimpose with Bs GpsB 5-64 Arg8 and Bs GpsB 5-64 Arg11 whereas molecule 2 accommodates Sp PBP2a Arg33 and Sp PBP2a Arg36 . c , d Close-up view of the interactions of Sp PBP2a from molecule 1 ( c ) and 2 ( d ) with Sp GpsB 4-63 . Key interfacial sidechains and backbone atoms are represented in stick format; Sp GpsB 4-63 is coloured cyan and Sp PBP2A 27-40 is coloured green. The van der Waals’ interactions between Sp GpsB Leu32 and Sp PBP1 Arg31 (molecule 1) and Sp PBP1 Arg33 (molecule 2) are in yellow. The carbonyl oxygens of Sp GpsB Ile11 , Sp GpsB Phe12 , Sp GpsB Glu13 and Sp GpsB Gln14 are denoted by respective red numerals

    Journal: Nature Communications

    Article Title: The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

    doi: 10.1038/s41467-018-08056-2

    Figure Lengend Snippet: The Sp PBP2a minidomain is not α-helical but still interacts with Sp GpsB through conserved arginines. a Arginine residues of Sp PBP2a play a key role in binding to Sp GpsB. Unless otherwise indicated, the fluorescence polarisation binding curves represent the interaction of TAMRA-labelled Sp PBP2a 23-45 peptides with wildtype Sp GpsB 1-63 . The relevant dissociation constants are listed in Supplementary Table 1 . b The structure of the Sp GpsB 4-63 : Sp PBP2a 27-40 complex reveals the critical role of Sp PBP2a arginines for the interaction with Sp GpsB. In this cartoon, Sp GpsB 4-63 is coloured cyan, and the Sp PBP2a 27-40 peptide is coloured yellow (molecule 1) and green (molecule 2). The sidechains of Arg8 and Arg11 from the Bs GpsB 5-64 : Bs PBP1 1-17 complex are shown as red sticks after a global superimposition of equivalent GpsB atoms. In molecule 1, Sp PBP2a Arg31 and Sp PBP2a Arg36 superimpose with Bs GpsB 5-64 Arg8 and Bs GpsB 5-64 Arg11 whereas molecule 2 accommodates Sp PBP2a Arg33 and Sp PBP2a Arg36 . c , d Close-up view of the interactions of Sp PBP2a from molecule 1 ( c ) and 2 ( d ) with Sp GpsB 4-63 . Key interfacial sidechains and backbone atoms are represented in stick format; Sp GpsB 4-63 is coloured cyan and Sp PBP2A 27-40 is coloured green. The van der Waals’ interactions between Sp GpsB Leu32 and Sp PBP1 Arg31 (molecule 1) and Sp PBP1 Arg33 (molecule 2) are in yellow. The carbonyl oxygens of Sp GpsB Ile11 , Sp GpsB Phe12 , Sp GpsB Glu13 and Sp GpsB Gln14 are denoted by respective red numerals

    Article Snippet: The fluorescein-labelled Bs YpbE1-21 and Sp PBP2b1-17 peptides used in FP assays were also synthesised chemically (Protein and Peptide Research Ltd); the Sp PBP2b1-17 peptide encompasses the first seventeen amino acids of Sp PBP2b from strain R6, based on the annotated sequence in the NCBI database (accession NP_359110 ). (iii) BsPBP1 proteins; Recombinant Bs PBP1 proteins were purified from E. coli membrane pellets resuspended in a buffer of 50 mM HEPES.NaOH pH 7.5, 500 mM NaCl, 3 mM MgCl2 , 0.3 mM DTT supplemented with Roche complete EDTA-free protease inhibitor cocktail, lysozyme and DNAase.

    Techniques: Binding Assay, Fluorescence

    The SRxxR(R/K) motif identifies Bs YpbE and Bs YrrS as new Bs GpsB binding partners. a , b Bs YpbE 1-18 and Bs YrrS 1-21 bind to Bs GpsB 1-68 at the same site as Bs PBP1. Fluorescence polarisation of Bs GpsB 1-68 binding to fluorescein-labelled Bs YpbE 1-18 ( a ) and fluorescein-labelled Bs YrrS 1-21 ( b ). The interaction of wild-type proteins is depicted by black curves, whereas the red curves and dashed black lines correspond to Bs GpsB 1-68 Asp31Ala and Bs GpsB 1-68 Tyr25Phe mutants, respectively. c BACTH reveals a new Bs GpsB interaction network involving proteins encoding the SRxxR(R/K) motif. The panel shows pairwise combinations of proteins expressed as N-terminal fusions to both halves of adenylate cyclase in the BACTH host strain. Their presence in complexes containing Bs RodZ, Bs PBP4b and Bs PBP1 imply roles for Bs YrrS and B sYpbE in sidewall synthesis during cell growth. The validity of the observed interactions is supported by the behaviour of the T18-GpsB 66-98 fusion, which does not interact with any other partner except T25-GpsB, and therefore acts as an internal control. The T18- Bs GpsB 66-98 :T25- Bs GpsB interaction is consistent with the hexameric nature of Bs GpsB 19 , 26 . The absence of interactions between Bs GpsB 66-98 and Bs YrrS or Bs YpbE is consistent with data in b that shows that the N-terminal domain of GpsB interacts with the SRxxR(R/K) motif of YpbE and YrrS. d A model recapitulates interactions between Bs GpsB and partners. Surface representations of the SAXS structure of Lm GpsB 26 (coloured teal) and the closest homologues in the PDB by sequence ( Bs PBP1, yellow, PDBid 2OLV ; Bs PBP4b, pale blue, 4L0L ; Bs RodZ, pink, 2WUS ; Bs MreC, dark grey, 2J5U ; Bs YpbE, salmon, 2MKX and the linker represents the disordered region, residues 131-188) are used as models for the B. subtilis proteins. Amorphous blobs, the surface area of which are scaled proportional to molecular weight, are used for Bs YrrS (green) and the extracellular domain of Bs RodZ (pink) where there is no structural information. The N-terminal domain of each membrane protein is cytoplasmic and 22-amino acid model helices represent each TM helix. The TMpred-predicted TM boundaries are: Bs PBP1 (38-60); Bs PBP4b (9-31); Bs YrrS (19-41); Bs YpbE (57-79); Bs RodZ (89-111) and Bs MreC (7-24). The GpsB-interacting domains of Bs YpbE, Bs YrrS and Bs PBP1 are based on the Bs PBP1 1-17 structure

    Journal: Nature Communications

    Article Title: The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

    doi: 10.1038/s41467-018-08056-2

    Figure Lengend Snippet: The SRxxR(R/K) motif identifies Bs YpbE and Bs YrrS as new Bs GpsB binding partners. a , b Bs YpbE 1-18 and Bs YrrS 1-21 bind to Bs GpsB 1-68 at the same site as Bs PBP1. Fluorescence polarisation of Bs GpsB 1-68 binding to fluorescein-labelled Bs YpbE 1-18 ( a ) and fluorescein-labelled Bs YrrS 1-21 ( b ). The interaction of wild-type proteins is depicted by black curves, whereas the red curves and dashed black lines correspond to Bs GpsB 1-68 Asp31Ala and Bs GpsB 1-68 Tyr25Phe mutants, respectively. c BACTH reveals a new Bs GpsB interaction network involving proteins encoding the SRxxR(R/K) motif. The panel shows pairwise combinations of proteins expressed as N-terminal fusions to both halves of adenylate cyclase in the BACTH host strain. Their presence in complexes containing Bs RodZ, Bs PBP4b and Bs PBP1 imply roles for Bs YrrS and B sYpbE in sidewall synthesis during cell growth. The validity of the observed interactions is supported by the behaviour of the T18-GpsB 66-98 fusion, which does not interact with any other partner except T25-GpsB, and therefore acts as an internal control. The T18- Bs GpsB 66-98 :T25- Bs GpsB interaction is consistent with the hexameric nature of Bs GpsB 19 , 26 . The absence of interactions between Bs GpsB 66-98 and Bs YrrS or Bs YpbE is consistent with data in b that shows that the N-terminal domain of GpsB interacts with the SRxxR(R/K) motif of YpbE and YrrS. d A model recapitulates interactions between Bs GpsB and partners. Surface representations of the SAXS structure of Lm GpsB 26 (coloured teal) and the closest homologues in the PDB by sequence ( Bs PBP1, yellow, PDBid 2OLV ; Bs PBP4b, pale blue, 4L0L ; Bs RodZ, pink, 2WUS ; Bs MreC, dark grey, 2J5U ; Bs YpbE, salmon, 2MKX and the linker represents the disordered region, residues 131-188) are used as models for the B. subtilis proteins. Amorphous blobs, the surface area of which are scaled proportional to molecular weight, are used for Bs YrrS (green) and the extracellular domain of Bs RodZ (pink) where there is no structural information. The N-terminal domain of each membrane protein is cytoplasmic and 22-amino acid model helices represent each TM helix. The TMpred-predicted TM boundaries are: Bs PBP1 (38-60); Bs PBP4b (9-31); Bs YrrS (19-41); Bs YpbE (57-79); Bs RodZ (89-111) and Bs MreC (7-24). The GpsB-interacting domains of Bs YpbE, Bs YrrS and Bs PBP1 are based on the Bs PBP1 1-17 structure

    Article Snippet: The fluorescein-labelled Bs YpbE1-21 and Sp PBP2b1-17 peptides used in FP assays were also synthesised chemically (Protein and Peptide Research Ltd); the Sp PBP2b1-17 peptide encompasses the first seventeen amino acids of Sp PBP2b from strain R6, based on the annotated sequence in the NCBI database (accession NP_359110 ). (iii) BsPBP1 proteins; Recombinant Bs PBP1 proteins were purified from E. coli membrane pellets resuspended in a buffer of 50 mM HEPES.NaOH pH 7.5, 500 mM NaCl, 3 mM MgCl2 , 0.3 mM DTT supplemented with Roche complete EDTA-free protease inhibitor cocktail, lysozyme and DNAase.

    Techniques: Binding Assay, Fluorescence, Sequencing, Molecular Weight

    Bs GpsB: Bs PBP1 interactions require conserved arginines in the α-helical cytoplasmic minidomain of Bs PBP1. a Bs GpsB interacts with the first 16 amino acids of Bs PBP1. SPR sensorgrams of full-length Bs GpsB against immobilised full-length Bs PBP1 (black) and Bs PBP1 17-914 (red). Bs GpsB does not interact with the Bs PBP1 17-914 coated chip, even when 25 μM GpsB is injected. b Cartoon (left) and surface representation (right) of the crystal structure of the Bs GpsB 5-64 : Bs PBP1 1-17 complex. Bs GpsB 5-64 is coloured cyan and Bs PBP1 1-17 is coloured green and the likely plane of the bacterial membrane is shown as a red box. c The Bs GpsB 5-64 : Bs PBP1 1-17 complex is dependent upon a conserved SRxxR(R/K) motif in Bs PBP1. Key interfacial residues in the Bs GpsB 5-64 : Bs PBP1 1-17 complex are shown as sticks and coloured (and labelled) blue and green, respectively. The carbonyl oxygens of Bs GpsB Ile13 , Bs GpsB Leu14 and Bs GpsB Lys16 are labelled with their respective red numerals. Hydrogen bonds are shown as black dashed lines and the van der Waals’ interactions between Bs GpsB Leu34 and Bs PBP1 Arg8 are in yellow. d Mutation of key Bs PBP1 interfacial residues in the structure of Bs GpsB 5-64 : Bs PBP1 1-17 leads to a loss of binding of position 16 TAMRA-labelled Bs PBP1 1-32 variants to Bs GpsB 1-68 as measured by fluorescence polarisation. When the Bs PBP1 1-32 peptide was labelled with fluorescein at position 31 the measured affinity was the same as if the fluorophore was at position 16, indicating that the fluorophore itself or its position in the peptide has no impact on the binding interaction. The calculated dissociation constants can be found in Supplementary Table 1

    Journal: Nature Communications

    Article Title: The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

    doi: 10.1038/s41467-018-08056-2

    Figure Lengend Snippet: Bs GpsB: Bs PBP1 interactions require conserved arginines in the α-helical cytoplasmic minidomain of Bs PBP1. a Bs GpsB interacts with the first 16 amino acids of Bs PBP1. SPR sensorgrams of full-length Bs GpsB against immobilised full-length Bs PBP1 (black) and Bs PBP1 17-914 (red). Bs GpsB does not interact with the Bs PBP1 17-914 coated chip, even when 25 μM GpsB is injected. b Cartoon (left) and surface representation (right) of the crystal structure of the Bs GpsB 5-64 : Bs PBP1 1-17 complex. Bs GpsB 5-64 is coloured cyan and Bs PBP1 1-17 is coloured green and the likely plane of the bacterial membrane is shown as a red box. c The Bs GpsB 5-64 : Bs PBP1 1-17 complex is dependent upon a conserved SRxxR(R/K) motif in Bs PBP1. Key interfacial residues in the Bs GpsB 5-64 : Bs PBP1 1-17 complex are shown as sticks and coloured (and labelled) blue and green, respectively. The carbonyl oxygens of Bs GpsB Ile13 , Bs GpsB Leu14 and Bs GpsB Lys16 are labelled with their respective red numerals. Hydrogen bonds are shown as black dashed lines and the van der Waals’ interactions between Bs GpsB Leu34 and Bs PBP1 Arg8 are in yellow. d Mutation of key Bs PBP1 interfacial residues in the structure of Bs GpsB 5-64 : Bs PBP1 1-17 leads to a loss of binding of position 16 TAMRA-labelled Bs PBP1 1-32 variants to Bs GpsB 1-68 as measured by fluorescence polarisation. When the Bs PBP1 1-32 peptide was labelled with fluorescein at position 31 the measured affinity was the same as if the fluorophore was at position 16, indicating that the fluorophore itself or its position in the peptide has no impact on the binding interaction. The calculated dissociation constants can be found in Supplementary Table 1

    Article Snippet: The fluorescein-labelled Bs YpbE1-21 and Sp PBP2b1-17 peptides used in FP assays were also synthesised chemically (Protein and Peptide Research Ltd); the Sp PBP2b1-17 peptide encompasses the first seventeen amino acids of Sp PBP2b from strain R6, based on the annotated sequence in the NCBI database (accession NP_359110 ). (iii) BsPBP1 proteins; Recombinant Bs PBP1 proteins were purified from E. coli membrane pellets resuspended in a buffer of 50 mM HEPES.NaOH pH 7.5, 500 mM NaCl, 3 mM MgCl2 , 0.3 mM DTT supplemented with Roche complete EDTA-free protease inhibitor cocktail, lysozyme and DNAase.

    Techniques: SPR Assay, Chromatin Immunoprecipitation, Injection, Mutagenesis, Binding Assay, Fluorescence