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  • 95
    Millipore pmsf
    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a <t>Sephadex</t> G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM <t>PMSF.</t> The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).
    Pmsf, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 24997 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 24997 article reviews
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    pmsf  (Abcam)
    90
    Abcam pmsf
    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a <t>Sephadex</t> G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM <t>PMSF.</t> The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).
    Pmsf, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc pmsf
    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a <t>Sephadex</t> G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM <t>PMSF.</t> The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).
    Pmsf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Amresco pmsf
    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a <t>Sephadex</t> G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM <t>PMSF.</t> The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).
    Pmsf, supplied by Amresco, used in various techniques. Bioz Stars score: 96/100, based on 324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pmsf - by Bioz Stars, 2020-02
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    96
    Applichem pmsf
    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a <t>Sephadex</t> G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM <t>PMSF.</t> The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).
    Pmsf, supplied by Applichem, used in various techniques. Bioz Stars score: 96/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pmsf - by Bioz Stars, 2020-02
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    96
    Beijing Solarbio Science pmsf
    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a <t>Sephadex</t> G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM <t>PMSF.</t> The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).
    Pmsf, supplied by Beijing Solarbio Science, used in various techniques. Bioz Stars score: 96/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Beyotime pmsf
    Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + l -carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with <t>RIPA</t> buffer supplemented with <t>PMSF</t> and then assessed with western blotting, and the expressions were normalized to control animals. ( A ) iNOS relative expression levels of ED19 chicken embryo hearts; ( B ) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group ( p
    Pmsf, supplied by Beyotime, used in various techniques. Bioz Stars score: 97/100, based on 3147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Boster Bio pmsf
    Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + l -carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with <t>RIPA</t> buffer supplemented with <t>PMSF</t> and then assessed with western blotting, and the expressions were normalized to control animals. ( A ) iNOS relative expression levels of ED19 chicken embryo hearts; ( B ) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group ( p
    Pmsf, supplied by Boster Bio, used in various techniques. Bioz Stars score: 96/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Carl Roth GmbH pmsf
    Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + l -carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with <t>RIPA</t> buffer supplemented with <t>PMSF</t> and then assessed with western blotting, and the expressions were normalized to control animals. ( A ) iNOS relative expression levels of ED19 chicken embryo hearts; ( B ) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group ( p
    Pmsf, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 96/100, based on 259 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Fisher Scientific pmsf
    Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + l -carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with <t>RIPA</t> buffer supplemented with <t>PMSF</t> and then assessed with western blotting, and the expressions were normalized to control animals. ( A ) iNOS relative expression levels of ED19 chicken embryo hearts; ( B ) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group ( p
    Pmsf, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 94/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    FUJIFILM pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 96/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Keygen Biotech pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 96/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pmsf - by Bioz Stars, 2020-02
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    96
    Merck KGaA pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 96/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Research Products International pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by Research Products International, used in various techniques. Bioz Stars score: 96/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pmsf  (Roche)
    99
    Roche pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 20991 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Santa Cruz Biotechnology pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 1061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Pmsf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 4324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pmsf  (3M Co)
    92
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    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Active Motif pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Bio Basic Canada pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Valiant pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Zhongshan Golden Bridge Company pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Epizyme pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    G Biosciences pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Beijing CWBio pmsf
    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), <t>PMSF</t> (1 mM) or <t>MG132</t> (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
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    Image Search Results


    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

    Journal: Cell motility and the cytoskeleton

    Article Title: The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

    doi: 10.1002/cm.20328

    Figure Lengend Snippet: The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

    Article Snippet: Sephadex G-50, N-ethylmaleimide (NEM), ATP, phalloidin, DTT and PMSF were purchased from Sigma Chemical Company (St. Louis, MO).

    Techniques: Sedimentation, SDS Page, Labeling, Construct

    Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + l -carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with RIPA buffer supplemented with PMSF and then assessed with western blotting, and the expressions were normalized to control animals. ( A ) iNOS relative expression levels of ED19 chicken embryo hearts; ( B ) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group ( p

    Journal: International Journal of Molecular Sciences

    Article Title: The Roles of Reactive Oxygen Species and Nitric Oxide in Perfluorooctanoic Acid-Induced Developmental Cardiotoxicity and l-Carnitine Mediated Protection

    doi: 10.3390/ijms18061229

    Figure Lengend Snippet: Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + l -carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with RIPA buffer supplemented with PMSF and then assessed with western blotting, and the expressions were normalized to control animals. ( A ) iNOS relative expression levels of ED19 chicken embryo hearts; ( B ) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group ( p

    Article Snippet: For iNOS, independent samples were extracted with a radio immunoprecipitation assay buffer (RIPA) supplemented with 1:100 PMSF (Beyotime, Beijing, China).

    Techniques: Western Blot, Injection, Incubation, Expressing

    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .

    Journal: Nature Communications

    Article Title: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17

    doi: 10.1038/ncomms15406

    Figure Lengend Snippet: Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .

    Article Snippet: Chemicals and antibodies Chemicals were obtained from the following sources: Hoechst 33342 (Thermo Fisher Scientific), PMSF (Wako chemicals), MG132 (Calbiochem), TRAIL (Merck Millipore), STS (Wako chemicals) and Percoll (Sigma-Aldrich).

    Techniques: Transfection, Plasmid Preparation, Fractionation, Mass Spectrometry, Incubation, Staining