protease inhibitor cocktail Search Results


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  • 99
    Thermo Fisher antiprotease inhibitor cocktail
    Antiprotease Inhibitor Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore antiprotease cocktail
    Antiprotease Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibitor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 110330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher antiprotease halt protease inhibitor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Antiprotease Halt Protease Inhibitor Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibitory cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitory Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibtor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibtor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protease inhibitor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitor Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protease inhibitors
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam protease inhibitor cocktails
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitor Cocktails, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega protease inhibitor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitor Cocktail, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore peptidase inhibitors
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Peptidase Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioVision protease inhibitor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitor Cocktail, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 1x protease inhibitors cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    1x Protease Inhibitors Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher complete protease inhibitor cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Complete Protease Inhibitor Cocktail, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bacterial protease inhibitors cocktail
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Bacterial Protease Inhibitors Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibitor cocktail tablets
    <t>PIC</t> treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007
    Protease Inhibitor Cocktail Tablets, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore complete protease inhibitor cocktail
    Aging-associated SIRT4 upregulation leads to a shifted L-OPA1 to S-OPA1 ratio in two different fibroblast senescence models Primary human dermal fibroblasts were either transfected with miR-15b inhibitors (or control oligonucleotides) or subjected to γ-irradiation (γIR; 20 Gy) (both in the presence or absence of siRNA duplexes against SIRT4) [ 35 ] followed by analysis of OPA1-L and OPA1-S expression by immunoblotting after four days ( A ) As a control for <t>complete</t> proteolytic processing of L-OPA1 to S-OPA1, fibroblasts were treated with CCCP (10 μM) for two hours. The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis in miR-15b inhibitor transfected fibroblasts ( B ) and cells subjected toγIR ( C ). To evaluate statistical significance (compared to control), two-way ANOVA followed by Tukey's test was performed (*p
    Complete Protease Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fresh protease inhibitors cocktail
    Aging-associated SIRT4 upregulation leads to a shifted L-OPA1 to S-OPA1 ratio in two different fibroblast senescence models Primary human dermal fibroblasts were either transfected with miR-15b inhibitors (or control oligonucleotides) or subjected to γ-irradiation (γIR; 20 Gy) (both in the presence or absence of siRNA duplexes against SIRT4) [ 35 ] followed by analysis of OPA1-L and OPA1-S expression by immunoblotting after four days ( A ) As a control for <t>complete</t> proteolytic processing of L-OPA1 to S-OPA1, fibroblasts were treated with CCCP (10 μM) for two hours. The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis in miR-15b inhibitor transfected fibroblasts ( B ) and cells subjected toγIR ( C ). To evaluate statistical significance (compared to control), two-way ANOVA followed by Tukey's test was performed (*p
    Fresh Protease Inhibitors Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PIC treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: PIC treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007

    Article Snippet: After 24 h, the medium was replaced with Optimem supplemented with 0.1% protease inhibitor cocktail (PIC) (P8340, Sigma-Aldrich, Saint Louis, MO), 0.1% DMSO, 0.1 mM AEBSF, 0.08 μM aprotinin, 1.4 μM E64, 4 μM bestatin, 2 μM leupeptin and 1.5 μM pepstatin A (Sigma-Aldrich).

    Techniques: Expressing, Immunofluorescence, Protease Inhibitor, Marker, Western Blot

    MMP13-mediated BACE1 regulation involves PI3K signalling and is unrelated to BACE1 transcription and protein degradation. ( A ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of the RTK inhibitor 341610 (4 μM). ( B ) SH-SY5Y cells were transfected with either control or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL) and 4 μM 341610. ( C ) SH-SY5Y cells were transfected with either control or MMP13 vector for 48 h in the absence or presence of 4 μM 341610, and PI3K activity was measured by ELISA. ( D ) p-Akt protein in SH-SY5Y cells transfected with either control or MMP13 vector for 48 h in the absence or presence of 4 μM 341610. ( E ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of the PI3K inhibitor LY294002 (LY, 5 and 10 μM). ( F ) SH-SY5Y cells were transfected with either control or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL) and LY294002 (LY, 5 and 10 μM). ( G ) Relative BACE1 mRNA levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of LY294002 (LY, 5 and 10 μM). ( H ) Relative Bace1 mRNA levels in HT22 cells treated with vehicle (CTRL) or Mmp13 shRNA-1 for 72 h. ( I and J ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of 1 μM MG132 (G) or 100 μM CQ (H). ( K ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of the transcriptional inhibitor actinomycin D (ActD, 0.1 μM) or the protein synthesis inhibitor cycloheximide (CHX, 5 μM). All values were normalized to CTRL (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; * P

    Journal: Brain

    Article Title: MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation

    doi: 10.1093/brain/awy305

    Figure Lengend Snippet: MMP13-mediated BACE1 regulation involves PI3K signalling and is unrelated to BACE1 transcription and protein degradation. ( A ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of the RTK inhibitor 341610 (4 μM). ( B ) SH-SY5Y cells were transfected with either control or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL) and 4 μM 341610. ( C ) SH-SY5Y cells were transfected with either control or MMP13 vector for 48 h in the absence or presence of 4 μM 341610, and PI3K activity was measured by ELISA. ( D ) p-Akt protein in SH-SY5Y cells transfected with either control or MMP13 vector for 48 h in the absence or presence of 4 μM 341610. ( E ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of the PI3K inhibitor LY294002 (LY, 5 and 10 μM). ( F ) SH-SY5Y cells were transfected with either control or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL) and LY294002 (LY, 5 and 10 μM). ( G ) Relative BACE1 mRNA levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of LY294002 (LY, 5 and 10 μM). ( H ) Relative Bace1 mRNA levels in HT22 cells treated with vehicle (CTRL) or Mmp13 shRNA-1 for 72 h. ( I and J ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of 1 μM MG132 (G) or 100 μM CQ (H). ( K ) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of the transcriptional inhibitor actinomycin D (ActD, 0.1 μM) or the protein synthesis inhibitor cycloheximide (CHX, 5 μM). All values were normalized to CTRL (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; * P

    Article Snippet: MMP13 inhibitors WAY170523 (Tocris Bioscience), CL82198 (Cayman) and SC205756 (Santa Cruz Biotechnology), FGF/PDGF/VEGF RTK inhibitor 341610 (Merk Millipore), PI3 kinase inhibitors LY294002 (Sigma Aldrich), eIF4E/eIF4G interaction inhibitor 4EGI1 (Selleck), protease inhibitor MG132 (Sigma), the transcriptional inhibitor actinomycin D (ActD, Sigma) and protein biosynthesis inhibitor cycloheximide (CHX, Sigma) were dissolved in dimethyl sulphoxide (DMSO) and lysosomal inhibitor chloroquine (CQ, Sigma) was dissolved in sterilized double-distilled H2 O.

    Techniques: Transfection, Plasmid Preparation, Activity Assay, Enzyme-linked Immunosorbent Assay, shRNA

    Western blot of exosome pellets. Exosomal proteins of interest including podocalyxin (PODXL), fibrocystin (FIBRO) and polycystin 1 (PC1) were examined by Western blot after storage and compared with freshly-isolated exosomes. These proteins were best preserved by the Sigma protease cocktail. a Polycystin 1 detection by Western blot was stable for 1 week at room temperature after exosomes were isolated and stored in 0.01 % sodium azide ( n = 7). However, polycystin 1 was not detected well from exosomes isolated after urine was frozen.at -80C probably because of precipitation issues and unlikely due to loss of protease activity. Effects of preservatives on Western blot of exosome pellet proteins PODXL, scramblase (PLSCR1), FIBRO, and smoothened (SMO). b Lane 1, Fresh, no preservatives; Lane 2, Fresh, no preservatives; Lane 3, −80 °C Frozen/Thawed; Lane 4, −80 °C Frozen/Thawed. c Comparison of varied amounts of protease inhibitors on exosome protein detection by Western blot. Initial assessment and following 6 months storage at −80 °C. Lane 1, 4.8 μL. Sigma protease inhibitor; Lane 2, 4.0 μL Sigma protease inhibitor; Lane 3, 1:100 Roche Complete tablet; Lane 4, −80 °C Frozen/Thawed; Lane 5, Fresh, no preservatives. d Exosomal Podocalyxin fared well at RT and at 4 °C. Sodium azide did not affect their survival. e Analysis of exosomes extracted from frozen (−80 °C) raw urine stored for 12 months

    Journal: BMC Nephrology

    Article Title: Strategy and rationale for urine collection protocols employed in the NEPTUNE study

    doi: 10.1186/s12882-015-0185-3

    Figure Lengend Snippet: Western blot of exosome pellets. Exosomal proteins of interest including podocalyxin (PODXL), fibrocystin (FIBRO) and polycystin 1 (PC1) were examined by Western blot after storage and compared with freshly-isolated exosomes. These proteins were best preserved by the Sigma protease cocktail. a Polycystin 1 detection by Western blot was stable for 1 week at room temperature after exosomes were isolated and stored in 0.01 % sodium azide ( n = 7). However, polycystin 1 was not detected well from exosomes isolated after urine was frozen.at -80C probably because of precipitation issues and unlikely due to loss of protease activity. Effects of preservatives on Western blot of exosome pellet proteins PODXL, scramblase (PLSCR1), FIBRO, and smoothened (SMO). b Lane 1, Fresh, no preservatives; Lane 2, Fresh, no preservatives; Lane 3, −80 °C Frozen/Thawed; Lane 4, −80 °C Frozen/Thawed. c Comparison of varied amounts of protease inhibitors on exosome protein detection by Western blot. Initial assessment and following 6 months storage at −80 °C. Lane 1, 4.8 μL. Sigma protease inhibitor; Lane 2, 4.0 μL Sigma protease inhibitor; Lane 3, 1:100 Roche Complete tablet; Lane 4, −80 °C Frozen/Thawed; Lane 5, Fresh, no preservatives. d Exosomal Podocalyxin fared well at RT and at 4 °C. Sodium azide did not affect their survival. e Analysis of exosomes extracted from frozen (−80 °C) raw urine stored for 12 months

    Article Snippet: There was substantially better detection of the podocyte marker of interest PODXL in cellular pellets stored with protease inhibitors, and again found that Sigma protease inhibitor product may in fact be superior to the more expensive Roche protease inhibitor cocktail (Additional file : Figure S1).

    Techniques: Western Blot, Isolation, Activity Assay, Protease Inhibitor

    Aging-associated SIRT4 upregulation leads to a shifted L-OPA1 to S-OPA1 ratio in two different fibroblast senescence models Primary human dermal fibroblasts were either transfected with miR-15b inhibitors (or control oligonucleotides) or subjected to γ-irradiation (γIR; 20 Gy) (both in the presence or absence of siRNA duplexes against SIRT4) [ 35 ] followed by analysis of OPA1-L and OPA1-S expression by immunoblotting after four days ( A ) As a control for complete proteolytic processing of L-OPA1 to S-OPA1, fibroblasts were treated with CCCP (10 μM) for two hours. The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis in miR-15b inhibitor transfected fibroblasts ( B ) and cells subjected toγIR ( C ). To evaluate statistical significance (compared to control), two-way ANOVA followed by Tukey's test was performed (*p

    Journal: Aging (Albany NY)

    Article Title: SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

    doi: 10.18632/aging.101307

    Figure Lengend Snippet: Aging-associated SIRT4 upregulation leads to a shifted L-OPA1 to S-OPA1 ratio in two different fibroblast senescence models Primary human dermal fibroblasts were either transfected with miR-15b inhibitors (or control oligonucleotides) or subjected to γ-irradiation (γIR; 20 Gy) (both in the presence or absence of siRNA duplexes against SIRT4) [ 35 ] followed by analysis of OPA1-L and OPA1-S expression by immunoblotting after four days ( A ) As a control for complete proteolytic processing of L-OPA1 to S-OPA1, fibroblasts were treated with CCCP (10 μM) for two hours. The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis in miR-15b inhibitor transfected fibroblasts ( B ) and cells subjected toγIR ( C ). To evaluate statistical significance (compared to control), two-way ANOVA followed by Tukey's test was performed (*p

    Article Snippet: Preparation of total cell lysates for immunoblot analysis Cleared cell lysates were generated using lysis buffer containing 0.3% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3 VO4 , 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4 O7 P2 , 1 μM DTT, 1x cOmplete™ protease inhibitor cocktail (CO-RO, Sigma-Aldrich).

    Techniques: Transfection, Irradiation, Expressing

    SIRT4-eGFP interacts with OPA1 ( A ) HEK293 cells stably expressing SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP were either untreated or treated with CCCP (10 μM, 2h) and thereafter subjected to OPA1 co-immunoprecipitation (IP) analysis using sepharose beads coupled anti-GFP single-domain-antibodies (nanobodies). Total cell lysates were loaded as input control (5%). CCCP treatment caused a complete proteolytic processing of L-OPA1 to S-OPA1. TACC3 was detected using specific antibodies and served as a representative negative co-immunoprecipitation control. ( B ) SIRT4 enzymatic activity is required for efficient interaction of SIRT4 with L-OPA1. The amount of L-OPA1 co-immunoprecipitated with SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP was determined in relation to the protein input and subjected to ImageJ-based densitometric analysis. To evaluate statistical significance, two-way ANOVA followed by Tukey's tests was performed (*p

    Journal: Aging (Albany NY)

    Article Title: SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

    doi: 10.18632/aging.101307

    Figure Lengend Snippet: SIRT4-eGFP interacts with OPA1 ( A ) HEK293 cells stably expressing SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP were either untreated or treated with CCCP (10 μM, 2h) and thereafter subjected to OPA1 co-immunoprecipitation (IP) analysis using sepharose beads coupled anti-GFP single-domain-antibodies (nanobodies). Total cell lysates were loaded as input control (5%). CCCP treatment caused a complete proteolytic processing of L-OPA1 to S-OPA1. TACC3 was detected using specific antibodies and served as a representative negative co-immunoprecipitation control. ( B ) SIRT4 enzymatic activity is required for efficient interaction of SIRT4 with L-OPA1. The amount of L-OPA1 co-immunoprecipitated with SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP was determined in relation to the protein input and subjected to ImageJ-based densitometric analysis. To evaluate statistical significance, two-way ANOVA followed by Tukey's tests was performed (*p

    Article Snippet: Preparation of total cell lysates for immunoblot analysis Cleared cell lysates were generated using lysis buffer containing 0.3% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3 VO4 , 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4 O7 P2 , 1 μM DTT, 1x cOmplete™ protease inhibitor cocktail (CO-RO, Sigma-Aldrich).

    Techniques: Stable Transfection, Expressing, Immunoprecipitation, Activity Assay

    SIRT4-eGFP expression stabilizes the mitochondrial fusion regulator L-OPA1 ( A ) The expression of L-OPA1 vs . S-OPA1 was analyzed by immunoblotting in HEK293 cells stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP. As a control for complete proteolytic processing of L-OPA1 to S-OPA1 eGFP-expressing control cells were treated with CCCP (10 μM) for two hours. ( B ) The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis. To evaluate statistical significance (compared to eGFP), two-way ANOVA followed by Tukey's tests was performed (**p

    Journal: Aging (Albany NY)

    Article Title: SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

    doi: 10.18632/aging.101307

    Figure Lengend Snippet: SIRT4-eGFP expression stabilizes the mitochondrial fusion regulator L-OPA1 ( A ) The expression of L-OPA1 vs . S-OPA1 was analyzed by immunoblotting in HEK293 cells stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(D28N)-eGFP. As a control for complete proteolytic processing of L-OPA1 to S-OPA1 eGFP-expressing control cells were treated with CCCP (10 μM) for two hours. ( B ) The ratio between the expression levels of L-OPA1 and S-OPA1 was determined by ImageJ-based densitometric analysis. To evaluate statistical significance (compared to eGFP), two-way ANOVA followed by Tukey's tests was performed (**p

    Article Snippet: Preparation of total cell lysates for immunoblot analysis Cleared cell lysates were generated using lysis buffer containing 0.3% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na3 VO4 , 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4 O7 P2 , 1 μM DTT, 1x cOmplete™ protease inhibitor cocktail (CO-RO, Sigma-Aldrich).

    Techniques: Expressing, Stable Transfection