actin ab 1  (Millipore)


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    Name:
    Anti Actin N terminal antibody
    Description:
    Actin is a cytoskeletal protein that regulates cell motility secretion phagocytosis and cytokinesis The NH2 terminal of actin may function as an antigen This terminal may also modulate actin interactions and may associate with proteins such as myosin
    Catalog Number:
    a2103
    Price:
    None
    Applications:
    Rabbit anti-actin, N-terminal antibody can be used for western blotting assays. The antibody can also be used for immunocytochemistry at 1-2μg/mL using cultured chicken fibroblasts. Furthermore, the antibody is suitable for use in immunohistochemistry at a concentration of 2-4μg/mL using sections of human appendix, mouse heart, and frog skeletal muscle.
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    Structured Review

    Millipore actin ab 1
    Anti Actin N terminal antibody
    Actin is a cytoskeletal protein that regulates cell motility secretion phagocytosis and cytokinesis The NH2 terminal of actin may function as an antigen This terminal may also modulate actin interactions and may associate with proteins such as myosin
    https://www.bioz.com/result/actin ab 1/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    actin ab 1 - by Bioz Stars, 2020-09
    96/100 stars

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    Related Articles

    Western Blot:

    Article Title: The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle
    Article Snippet: .. This was initially probed for embryonic myosin heavy chain, then stripped (Restore Western blot stripping buffer, Thermo Scientific) and probed for anti-α-actin (A2103, Sigma-Aldrich). ..

    Article Title: Temporal and spatial characterization of nonsense-mediated mRNA decay
    Article Snippet: .. Anti-RENT1 UPF1 antibody (Bethyl Laboratories, A300-036A) was used for Western Blot ( ). β-Actin, detected by rabbit anti-actin antibody (Sigma, A2103) was used as a loading control. ..

    Incubation:

    Article Title: A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans
    Article Snippet: .. The membrane was placed in blocking buffer (5% fat-free milk in TBS-Tween 20 buffer) overnight and incubated for 1 hour with polyclonal anti-GFP (Life Technologies cat. A21311) or anti-actin antibody (Sigma cat. A2103), which was used as the loading control, at a dilution of 1:500 or 1:2000, respectively, and an appropriate secondary antibody for 1 hour. ..

    Stripping:

    Article Title: The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle
    Article Snippet: .. This was initially probed for embryonic myosin heavy chain, then stripped (Restore Western blot stripping buffer, Thermo Scientific) and probed for anti-α-actin (A2103, Sigma-Aldrich). ..

    Blocking Assay:

    Article Title: A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans
    Article Snippet: .. The membrane was placed in blocking buffer (5% fat-free milk in TBS-Tween 20 buffer) overnight and incubated for 1 hour with polyclonal anti-GFP (Life Technologies cat. A21311) or anti-actin antibody (Sigma cat. A2103), which was used as the loading control, at a dilution of 1:500 or 1:2000, respectively, and an appropriate secondary antibody for 1 hour. ..

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  • 93
    Millipore anti dectin 1
    Gene expression and translation of <t>dectin-1</t> signaling proteins. a Illustration of selected signaling molecules downstream of dectin-1; b Polysome profiles and c quantification of signalosome genes (qPCR) in monosome, disome, and light and heavy polysome fractions (monocytes). Data are from 4 subjects per age group (boxes and whiskers; RQ = relative quantification); d Quantification of signalosome genes (qPCR) in total RNA fractions (4 to 5 subjects/age group; mean ± SD); e Surface expression of dectin-1 (flow cytometry, mononuclear cells, gated on CD14-expressing cells; data pooled from 10 to 23 subjects per age group; boxes and whiskers); f Representative (cropped) Western blot of MALT1 and Bcl10 protein expression in monocytes after 0 to 60 min LPS stimulation. Representative blot is from a 29 weeks gestation sample. Images cropped from same blot probes with each antibody; cumulative quantification of 4 independent Western blot experiments for g MALT1 and h Bcl10 (mean ± SD)
    Anti Dectin 1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti dectin 1/product/Millipore
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti dectin 1 - by Bioz Stars, 2020-09
    93/100 stars
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    80
    Millipore rabbit anti flash
    Activation of PR transgenes by wild type histone gene arrays A-1 to A-8) Intact salivary gland nuclei from 3 rd instar larvae of the indicated genotypes, all of which contained HisC and stained for the HLB components <t>Mxc</t> and <t>FLASH.</t> Ectopic HLBs are indicated by arrows and dashed boxes, and were present in all genotypes except the 12x PR . Scale bar 10 microns. B) Quantification of the percentage of ectopic HLBs in the salivary gland nuclei in panel B. C) Schematic of the assay used to distinguish 12x PR from 12x HWT cDNA. RT-PCR amplification of H3 mRNA from 5-7 hr old embryos of the indicated genotypes, followed by digestion with SacI and visualized on an 8% polyacrylamide gel. D) RT-PCR amplification of H2a mRNA from larvae of indicated genotypes, followed by digestion with XhoI and visualization on an agarose gel. E) Top: Schematic of the BAC-based PR-1 histone array containing 11 PR and 1 DWT repeat unit. Bottom: Analysis of BAC constructs containing WT and PR repeats using PCR as in panel 1B and C.
    Rabbit Anti Flash, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti flash/product/Millipore
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    88
    Millipore anti ns1 antibody
    Secretory phenotype of Gaussia Luciferase after insertion of the <t>DENV-NS1</t> CBD. A. Schematic representation of the construction of G Luc with the inserted DENV-NS1 CBD. Color boxes indicate the origin of the CBD. Ac5: Actin5C promoter; Cherry: red fluorescent protein; neo: geneticin resistance; SS: signal secretion, catalytic domain of luciferase. B. Confocal microscopy analysis of GLuc chimeras with CAV-1. Recombinant GLuc chimeras were transfected and 24hpt, cells were probed for G Luc (shown in green) and CAV-1(shown in red). The Pearson correlation coefficients (PCC) measured in at least 20 confocal independent images with 0.41 μM laser sections for each condition are shown. C. Secretion of Gaussia Lucifearse chimeras (DENV2 and YFV) in C6/36 cells treated for 48 hrs with BFA, or DMSO as control. Luciferase activity in control cells was taken as 100%. Experiments are based on at least three independent experiments with each chimera ± standard error; significant differences compared with controls are denoted by *( p
    Anti Ns1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ns1 antibody/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ns1 antibody - by Bioz Stars, 2020-09
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    88
    Millipore rabbit anti human mmp 1
    <t>MMP-1</t> expression in Bowes RGP cells promotes tumor growth and metastasis. ( a ) Bowes, Bowes-pCMV and Bowes-pCMV-MMP1 cells were injected intradermally into nude mice (10 6 cells/injection). Tumor incidence was noted (table) and tumors were measured weekly with calipers. * p
    Rabbit Anti Human Mmp 1, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti human mmp 1/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti human mmp 1 - by Bioz Stars, 2020-09
    88/100 stars
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    Image Search Results


    Gene expression and translation of dectin-1 signaling proteins. a Illustration of selected signaling molecules downstream of dectin-1; b Polysome profiles and c quantification of signalosome genes (qPCR) in monosome, disome, and light and heavy polysome fractions (monocytes). Data are from 4 subjects per age group (boxes and whiskers; RQ = relative quantification); d Quantification of signalosome genes (qPCR) in total RNA fractions (4 to 5 subjects/age group; mean ± SD); e Surface expression of dectin-1 (flow cytometry, mononuclear cells, gated on CD14-expressing cells; data pooled from 10 to 23 subjects per age group; boxes and whiskers); f Representative (cropped) Western blot of MALT1 and Bcl10 protein expression in monocytes after 0 to 60 min LPS stimulation. Representative blot is from a 29 weeks gestation sample. Images cropped from same blot probes with each antibody; cumulative quantification of 4 independent Western blot experiments for g MALT1 and h Bcl10 (mean ± SD)

    Journal: Nature Communications

    Article Title: Cellular metabolism constrains innate immune responses in early human ontogeny

    doi: 10.1038/s41467-018-07215-9

    Figure Lengend Snippet: Gene expression and translation of dectin-1 signaling proteins. a Illustration of selected signaling molecules downstream of dectin-1; b Polysome profiles and c quantification of signalosome genes (qPCR) in monosome, disome, and light and heavy polysome fractions (monocytes). Data are from 4 subjects per age group (boxes and whiskers; RQ = relative quantification); d Quantification of signalosome genes (qPCR) in total RNA fractions (4 to 5 subjects/age group; mean ± SD); e Surface expression of dectin-1 (flow cytometry, mononuclear cells, gated on CD14-expressing cells; data pooled from 10 to 23 subjects per age group; boxes and whiskers); f Representative (cropped) Western blot of MALT1 and Bcl10 protein expression in monocytes after 0 to 60 min LPS stimulation. Representative blot is from a 29 weeks gestation sample. Images cropped from same blot probes with each antibody; cumulative quantification of 4 independent Western blot experiments for g MALT1 and h Bcl10 (mean ± SD)

    Article Snippet: For blocking experiments, MNCs were pre-incubated for 1 h at 37 °C with neutralizing anti-dectin-1 or anti-dectin-2 antibodies, mepazine hydrochloride (10 µM, EMD Millipore #5005000001), cycloheximide (100 μg ml-1 , Sigma-Aldrich #C4859), ST 2825 (a MyD88 inhibitor, 10 µM, AdooQ Bioscience, #A15248-1), GW5074 (a Raf-1 inhibitor, 1 µM, Cayman Chemical #10010368-1), Piceatannol (a Syk inhibitor, 20 µM, Cayman Chemical, #10009355-5).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot

    MALT1 is essential for Candida recognition and curdlan signaling in human monocytes. IL-1β production after stimulation (24 h) with a curdlan or b LPS, or with c C. albicans or d C. parapsilosis , and upon inhibition (i) of MALT1 (using mepazine hydrochloride), MyD88 (ST2825), Raf (GW5074) or Syk (Piceatannol), or blocking of dectin-1 or dectin-2 using antibodies (data pooled from 6 experiments (6 subjects); mean ± SD); e Effect of MALT1 inhibition, dectin-1-blocking antibody or actin polymerization inhibition (using cytochalasin- d ) on phagocytosis of Candida by monocytes (boxes and whiskers; data also from 6 experiments). Statistical significance was calculated using 2-sided paired t -tests

    Journal: Nature Communications

    Article Title: Cellular metabolism constrains innate immune responses in early human ontogeny

    doi: 10.1038/s41467-018-07215-9

    Figure Lengend Snippet: MALT1 is essential for Candida recognition and curdlan signaling in human monocytes. IL-1β production after stimulation (24 h) with a curdlan or b LPS, or with c C. albicans or d C. parapsilosis , and upon inhibition (i) of MALT1 (using mepazine hydrochloride), MyD88 (ST2825), Raf (GW5074) or Syk (Piceatannol), or blocking of dectin-1 or dectin-2 using antibodies (data pooled from 6 experiments (6 subjects); mean ± SD); e Effect of MALT1 inhibition, dectin-1-blocking antibody or actin polymerization inhibition (using cytochalasin- d ) on phagocytosis of Candida by monocytes (boxes and whiskers; data also from 6 experiments). Statistical significance was calculated using 2-sided paired t -tests

    Article Snippet: For blocking experiments, MNCs were pre-incubated for 1 h at 37 °C with neutralizing anti-dectin-1 or anti-dectin-2 antibodies, mepazine hydrochloride (10 µM, EMD Millipore #5005000001), cycloheximide (100 μg ml-1 , Sigma-Aldrich #C4859), ST 2825 (a MyD88 inhibitor, 10 µM, AdooQ Bioscience, #A15248-1), GW5074 (a Raf-1 inhibitor, 1 µM, Cayman Chemical #10010368-1), Piceatannol (a Syk inhibitor, 20 µM, Cayman Chemical, #10009355-5).

    Techniques: Inhibition, Blocking Assay

    Impaired glycolysis and mTOR activity in preterm neonatal monocytes. a Effect of blocking glycolysis on cytokine responses to LPS stimulation (24 h) in mononuclear cells (2-sided paired t -tests; boxes and whiskers; 4 to 9 samples per condition; 2-DG = 2-deoxy-d-glucose); b Extracellular acidification rates (ECAR, normalized to total protein content), under baseline glucose-free conditions, after addition of glucose, oligomycin, and 2-DG (representative experiment from a 29 week gestation preterm sample); c Glycolytic capacity (cumulative data pooled from 4 independent experiments; 3–9 subjects per age group; mean ± SD; p value by Mann–Whitney U test); d Lactate production in mononuclear cells after LPS stimulation (24 h); p value = effect of age by 2-way ANOVA. Data from 9 to 10 subjects per age group; boxes and whiskers; e Effect of blocking glycolysis or translation (using cycloheximide, CHX) on the phagocytosis of C. albicans (C a ); boxes and whiskers; 3 subjects; Cyto-D = cytochalasin-D; f Depiction of signaling events between Toll-like receptor and Raf-1-mediated dectin-1 activation, mTOR phosphorylation, and increased glycolysis and protein synthesis; g Western blot image (cropped) of mTOR/4EBP1 expression and mTOR phosphorylation (monocytes; preterm sample from 29 weeks gestation; cropped images from same blot probed for mTOR, β-actin, and 4EBP1, and stripped/re-probed for phospho-mTOR); h Expression of mTOR-related genes (monocytes; 6–12 per age group; boxes and whiskers; 2-way ANOVA across age groups, only significant p values are shown)

    Journal: Nature Communications

    Article Title: Cellular metabolism constrains innate immune responses in early human ontogeny

    doi: 10.1038/s41467-018-07215-9

    Figure Lengend Snippet: Impaired glycolysis and mTOR activity in preterm neonatal monocytes. a Effect of blocking glycolysis on cytokine responses to LPS stimulation (24 h) in mononuclear cells (2-sided paired t -tests; boxes and whiskers; 4 to 9 samples per condition; 2-DG = 2-deoxy-d-glucose); b Extracellular acidification rates (ECAR, normalized to total protein content), under baseline glucose-free conditions, after addition of glucose, oligomycin, and 2-DG (representative experiment from a 29 week gestation preterm sample); c Glycolytic capacity (cumulative data pooled from 4 independent experiments; 3–9 subjects per age group; mean ± SD; p value by Mann–Whitney U test); d Lactate production in mononuclear cells after LPS stimulation (24 h); p value = effect of age by 2-way ANOVA. Data from 9 to 10 subjects per age group; boxes and whiskers; e Effect of blocking glycolysis or translation (using cycloheximide, CHX) on the phagocytosis of C. albicans (C a ); boxes and whiskers; 3 subjects; Cyto-D = cytochalasin-D; f Depiction of signaling events between Toll-like receptor and Raf-1-mediated dectin-1 activation, mTOR phosphorylation, and increased glycolysis and protein synthesis; g Western blot image (cropped) of mTOR/4EBP1 expression and mTOR phosphorylation (monocytes; preterm sample from 29 weeks gestation; cropped images from same blot probed for mTOR, β-actin, and 4EBP1, and stripped/re-probed for phospho-mTOR); h Expression of mTOR-related genes (monocytes; 6–12 per age group; boxes and whiskers; 2-way ANOVA across age groups, only significant p values are shown)

    Article Snippet: For blocking experiments, MNCs were pre-incubated for 1 h at 37 °C with neutralizing anti-dectin-1 or anti-dectin-2 antibodies, mepazine hydrochloride (10 µM, EMD Millipore #5005000001), cycloheximide (100 μg ml-1 , Sigma-Aldrich #C4859), ST 2825 (a MyD88 inhibitor, 10 µM, AdooQ Bioscience, #A15248-1), GW5074 (a Raf-1 inhibitor, 1 µM, Cayman Chemical #10010368-1), Piceatannol (a Syk inhibitor, 20 µM, Cayman Chemical, #10009355-5).

    Techniques: Activity Assay, Blocking Assay, MANN-WHITNEY, Activation Assay, Western Blot, Expressing

    Importance of dectin-1 in response to Candida in neonatal monocytes. a Antibody blocking of dectin-1 reduces cytokine production to C. albicans (C a ) in adult mononuclear cells (one-sided t -test with Welch’s correction for unequal variance; 6 to 12 subjects per condition; boxes and whiskers); b Phagocytosis of C. albicans upon blocking with anti-dectin-1 receptor antibody, same-isotype control or cytochalasin-D (Cyto-D); (11 to 18 subjects per age group; boxes and whiskers); Blocking of c C. albicans (C a ) or d C. parapsilosis (C p ) phagocytosis using laminarin (blocking dectin-1), mannan (blocking dectin-2 and mannose receptor), anti-DC-SIGN, and anti-CD206 antibodies, or a combination of all three antibodies (bar graph with mean ± standard deviation (SD); 2 to 3 subjects per age group)

    Journal: Nature Communications

    Article Title: Cellular metabolism constrains innate immune responses in early human ontogeny

    doi: 10.1038/s41467-018-07215-9

    Figure Lengend Snippet: Importance of dectin-1 in response to Candida in neonatal monocytes. a Antibody blocking of dectin-1 reduces cytokine production to C. albicans (C a ) in adult mononuclear cells (one-sided t -test with Welch’s correction for unequal variance; 6 to 12 subjects per condition; boxes and whiskers); b Phagocytosis of C. albicans upon blocking with anti-dectin-1 receptor antibody, same-isotype control or cytochalasin-D (Cyto-D); (11 to 18 subjects per age group; boxes and whiskers); Blocking of c C. albicans (C a ) or d C. parapsilosis (C p ) phagocytosis using laminarin (blocking dectin-1), mannan (blocking dectin-2 and mannose receptor), anti-DC-SIGN, and anti-CD206 antibodies, or a combination of all three antibodies (bar graph with mean ± standard deviation (SD); 2 to 3 subjects per age group)

    Article Snippet: For blocking experiments, MNCs were pre-incubated for 1 h at 37 °C with neutralizing anti-dectin-1 or anti-dectin-2 antibodies, mepazine hydrochloride (10 µM, EMD Millipore #5005000001), cycloheximide (100 μg ml-1 , Sigma-Aldrich #C4859), ST 2825 (a MyD88 inhibitor, 10 µM, AdooQ Bioscience, #A15248-1), GW5074 (a Raf-1 inhibitor, 1 µM, Cayman Chemical #10010368-1), Piceatannol (a Syk inhibitor, 20 µM, Cayman Chemical, #10009355-5).

    Techniques: Blocking Assay, Standard Deviation

    Activation of PR transgenes by wild type histone gene arrays A-1 to A-8) Intact salivary gland nuclei from 3 rd instar larvae of the indicated genotypes, all of which contained HisC and stained for the HLB components Mxc and FLASH. Ectopic HLBs are indicated by arrows and dashed boxes, and were present in all genotypes except the 12x PR . Scale bar 10 microns. B) Quantification of the percentage of ectopic HLBs in the salivary gland nuclei in panel B. C) Schematic of the assay used to distinguish 12x PR from 12x HWT cDNA. RT-PCR amplification of H3 mRNA from 5-7 hr old embryos of the indicated genotypes, followed by digestion with SacI and visualized on an 8% polyacrylamide gel. D) RT-PCR amplification of H2a mRNA from larvae of indicated genotypes, followed by digestion with XhoI and visualization on an agarose gel. E) Top: Schematic of the BAC-based PR-1 histone array containing 11 PR and 1 DWT repeat unit. Bottom: Analysis of BAC constructs containing WT and PR repeats using PCR as in panel 1B and C.

    Journal: bioRxiv

    Article Title: Drosophila Histone Locus Body assembly and function involves multiple interactions

    doi: 10.1101/2020.03.16.994483

    Figure Lengend Snippet: Activation of PR transgenes by wild type histone gene arrays A-1 to A-8) Intact salivary gland nuclei from 3 rd instar larvae of the indicated genotypes, all of which contained HisC and stained for the HLB components Mxc and FLASH. Ectopic HLBs are indicated by arrows and dashed boxes, and were present in all genotypes except the 12x PR . Scale bar 10 microns. B) Quantification of the percentage of ectopic HLBs in the salivary gland nuclei in panel B. C) Schematic of the assay used to distinguish 12x PR from 12x HWT cDNA. RT-PCR amplification of H3 mRNA from 5-7 hr old embryos of the indicated genotypes, followed by digestion with SacI and visualized on an 8% polyacrylamide gel. D) RT-PCR amplification of H2a mRNA from larvae of indicated genotypes, followed by digestion with XhoI and visualization on an agarose gel. E) Top: Schematic of the BAC-based PR-1 histone array containing 11 PR and 1 DWT repeat unit. Bottom: Analysis of BAC constructs containing WT and PR repeats using PCR as in panel 1B and C.

    Article Snippet: Immunofluorescence We used primary antibodies at the following concentrations: rabbit anti-CLAMP (1:1000), guinea pig anti-Mxc (1:2000), guinea pig anti-Mute (1:5000), rabbit anti-FLASH (1:2000), rabbit anti-Lsm10 (1:1000), mouse anti-MPM-2 (1:100; Millipore), rabbit anti-GAF (1:1000), mouse anti-LacI (1:1000; Millipore).

    Techniques: Activation Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, BAC Assay, Construct, Polymerase Chain Reaction

    The PR transgene is expressed and forms an HLB in the absence of the endogenous RD histone genes. A, B) RNA was isolated from 5-7 hr embryos (A) or 3 rd instar larvae (B) containing the 12x DWT or 12x PR transgene in the presence or absence of HisC , with wild-type embryos as a control (lane 1). RT-PCR products using H2a primers were digested with Xho I and visualized using a 1.5% agarose gel (which does not resolve the two restriction fragments from the endogenous genes as in Fig. 1E ). C, D) Polytene chromosomes from HisC deletion homozygous 3 rd instar larval salivary glands rescued by either the 12x DWT or 12x PR transgene stained with antibodies against HLB components Lsm11 and FLASH (C) or Mxc and Mute (D). E, F) Total RNA from 0-3h or 3-5h embryos (D) or 3 rd instar larvae (E) from wild-type (yw) or HisC deletion homozygotes containing either the 12x DWT or 12x PR transgene analyzed by Northern blotting using a radiolabeled H3 coding region probe. * = normally processed H3 mRNA. A FLASH mutant that cannot recruit the processing machinery to the HLB ( Tatomer et al ., 2016b ) was used as a positive control for production of poly(A)+ histone mRNA. G) Syncytial, embryos from HisC deletion homozygous stocks rescued by 12x DWT or 12x PR transgenes and stained with Mxc to monitor HLB formation in development. 3 consecutive syncytial nuclear cycles (top to bottom) were analyzed.

    Journal: bioRxiv

    Article Title: Drosophila Histone Locus Body assembly and function involves multiple interactions

    doi: 10.1101/2020.03.16.994483

    Figure Lengend Snippet: The PR transgene is expressed and forms an HLB in the absence of the endogenous RD histone genes. A, B) RNA was isolated from 5-7 hr embryos (A) or 3 rd instar larvae (B) containing the 12x DWT or 12x PR transgene in the presence or absence of HisC , with wild-type embryos as a control (lane 1). RT-PCR products using H2a primers were digested with Xho I and visualized using a 1.5% agarose gel (which does not resolve the two restriction fragments from the endogenous genes as in Fig. 1E ). C, D) Polytene chromosomes from HisC deletion homozygous 3 rd instar larval salivary glands rescued by either the 12x DWT or 12x PR transgene stained with antibodies against HLB components Lsm11 and FLASH (C) or Mxc and Mute (D). E, F) Total RNA from 0-3h or 3-5h embryos (D) or 3 rd instar larvae (E) from wild-type (yw) or HisC deletion homozygotes containing either the 12x DWT or 12x PR transgene analyzed by Northern blotting using a radiolabeled H3 coding region probe. * = normally processed H3 mRNA. A FLASH mutant that cannot recruit the processing machinery to the HLB ( Tatomer et al ., 2016b ) was used as a positive control for production of poly(A)+ histone mRNA. G) Syncytial, embryos from HisC deletion homozygous stocks rescued by 12x DWT or 12x PR transgenes and stained with Mxc to monitor HLB formation in development. 3 consecutive syncytial nuclear cycles (top to bottom) were analyzed.

    Article Snippet: Immunofluorescence We used primary antibodies at the following concentrations: rabbit anti-CLAMP (1:1000), guinea pig anti-Mxc (1:2000), guinea pig anti-Mute (1:5000), rabbit anti-FLASH (1:2000), rabbit anti-Lsm10 (1:1000), mouse anti-MPM-2 (1:100; Millipore), rabbit anti-GAF (1:1000), mouse anti-LacI (1:1000; Millipore).

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Northern Blot, Mutagenesis, Positive Control

    Secretory phenotype of Gaussia Luciferase after insertion of the DENV-NS1 CBD. A. Schematic representation of the construction of G Luc with the inserted DENV-NS1 CBD. Color boxes indicate the origin of the CBD. Ac5: Actin5C promoter; Cherry: red fluorescent protein; neo: geneticin resistance; SS: signal secretion, catalytic domain of luciferase. B. Confocal microscopy analysis of GLuc chimeras with CAV-1. Recombinant GLuc chimeras were transfected and 24hpt, cells were probed for G Luc (shown in green) and CAV-1(shown in red). The Pearson correlation coefficients (PCC) measured in at least 20 confocal independent images with 0.41 μM laser sections for each condition are shown. C. Secretion of Gaussia Lucifearse chimeras (DENV2 and YFV) in C6/36 cells treated for 48 hrs with BFA, or DMSO as control. Luciferase activity in control cells was taken as 100%. Experiments are based on at least three independent experiments with each chimera ± standard error; significant differences compared with controls are denoted by *( p

    Journal: bioRxiv

    Article Title: The secretory fate of flavivirus NS1 in mosquito cells is influenced by the caveolin binding domain

    doi: 10.1101/2019.12.16.879031

    Figure Lengend Snippet: Secretory phenotype of Gaussia Luciferase after insertion of the DENV-NS1 CBD. A. Schematic representation of the construction of G Luc with the inserted DENV-NS1 CBD. Color boxes indicate the origin of the CBD. Ac5: Actin5C promoter; Cherry: red fluorescent protein; neo: geneticin resistance; SS: signal secretion, catalytic domain of luciferase. B. Confocal microscopy analysis of GLuc chimeras with CAV-1. Recombinant GLuc chimeras were transfected and 24hpt, cells were probed for G Luc (shown in green) and CAV-1(shown in red). The Pearson correlation coefficients (PCC) measured in at least 20 confocal independent images with 0.41 μM laser sections for each condition are shown. C. Secretion of Gaussia Lucifearse chimeras (DENV2 and YFV) in C6/36 cells treated for 48 hrs with BFA, or DMSO as control. Luciferase activity in control cells was taken as 100%. Experiments are based on at least three independent experiments with each chimera ± standard error; significant differences compared with controls are denoted by *( p

    Article Snippet: Measurement of secreted NS1 protein The presence of flavivirus NS1 in cell supernatants was measured using a non-commercial, in-house, ELISA.

    Techniques: Luciferase, Confocal Microscopy, Recombinant, Transfection, Periodic Counter-current Chromatography, Activity Assay

    Secretory phenotype of DENV2-NS1 after addition of the signal peptide of Gaussia Luciferase. A. Schematic representation of the construction obtained after insertion of the G Luc signal peptide in the N-terminal of recombinant DENV2-NS1. The sequence MGVKVLFALICIAVAEA was introduced by site directed mutagenesis as described in the methods section. B. Secretion of GLuc-NS1 was measured in cells supernatants 48h post transfection. C. Co-localization between DENV2-NS1-wt or DENV2-GLuc-NS1 and ER (GRP78) and cis -Golgi (GM130) markers. Recombinants NS1 were transfected and 24hpt, cells were probed for NS1 (shown in green) and GRP78 or GM130 (shown in red). D. Pearson correlation coefficients (PCC) for organelle-NS1 were measured in at least 20 confocal independent images with 0.48 μM laser sections. The bars represent means ± standard error. Data was evaluated using the 2way ANOVA test and significant differences are denoted by *( p ≤0.05).

    Journal: bioRxiv

    Article Title: The secretory fate of flavivirus NS1 in mosquito cells is influenced by the caveolin binding domain

    doi: 10.1101/2019.12.16.879031

    Figure Lengend Snippet: Secretory phenotype of DENV2-NS1 after addition of the signal peptide of Gaussia Luciferase. A. Schematic representation of the construction obtained after insertion of the G Luc signal peptide in the N-terminal of recombinant DENV2-NS1. The sequence MGVKVLFALICIAVAEA was introduced by site directed mutagenesis as described in the methods section. B. Secretion of GLuc-NS1 was measured in cells supernatants 48h post transfection. C. Co-localization between DENV2-NS1-wt or DENV2-GLuc-NS1 and ER (GRP78) and cis -Golgi (GM130) markers. Recombinants NS1 were transfected and 24hpt, cells were probed for NS1 (shown in green) and GRP78 or GM130 (shown in red). D. Pearson correlation coefficients (PCC) for organelle-NS1 were measured in at least 20 confocal independent images with 0.48 μM laser sections. The bars represent means ± standard error. Data was evaluated using the 2way ANOVA test and significant differences are denoted by *( p ≤0.05).

    Article Snippet: Measurement of secreted NS1 protein The presence of flavivirus NS1 in cell supernatants was measured using a non-commercial, in-house, ELISA.

    Techniques: Luciferase, Recombinant, Sequencing, Mutagenesis, Transfection, Periodic Counter-current Chromatography

    Secretory phenotype of CBD-chimeras of flavivirus NS1. A. Schematic representation of the chimeras generated from DENV (left) and YFV (right) NS1. Colors indicate the origin of the CBD. B. Secretion of DENV2-NS1 chimeras with ZIKV and YFV CBD. C. Secretion of YFV-NS1 chimeras with DENV and WNV CBD. Twenty-four hours post-transfection, C6/36 cells were treated with DMSO (control) or with 25 µM BFA and the supernatants harvested after 48 h. Levels of secreted NS1 were measured by ELISA. Data are mean of at least 3 independent experiments ± standard error; significant differences compared with controls are denoted by *( p

    Journal: bioRxiv

    Article Title: The secretory fate of flavivirus NS1 in mosquito cells is influenced by the caveolin binding domain

    doi: 10.1101/2019.12.16.879031

    Figure Lengend Snippet: Secretory phenotype of CBD-chimeras of flavivirus NS1. A. Schematic representation of the chimeras generated from DENV (left) and YFV (right) NS1. Colors indicate the origin of the CBD. B. Secretion of DENV2-NS1 chimeras with ZIKV and YFV CBD. C. Secretion of YFV-NS1 chimeras with DENV and WNV CBD. Twenty-four hours post-transfection, C6/36 cells were treated with DMSO (control) or with 25 µM BFA and the supernatants harvested after 48 h. Levels of secreted NS1 were measured by ELISA. Data are mean of at least 3 independent experiments ± standard error; significant differences compared with controls are denoted by *( p

    Article Snippet: Measurement of secreted NS1 protein The presence of flavivirus NS1 in cell supernatants was measured using a non-commercial, in-house, ELISA.

    Techniques: Generated, Transfection, Enzyme-linked Immunosorbent Assay

    Schematic representation of the overall site directed mutagenesis strategy. 1. Generation of recombinant flavivirus NS1. The details for the construction of recombinants NS1 are in Materials and Methods. Each construct was used as template for site directed mutagenesis. Actin5C promoter; cherry, red fluorescent protein; eGFP, eukaryotic green fluorescent protein; neo, resistance to neomycin, kanamycin and G418, each gene separated by a T2A peptide. Yellow and blue; DENV and ZIKV NS1 CBD sequences; red; YFV NS1 incomplete CDB. 2. Substitutions of aromatic residues in CBD to Ala or Thr. CBD sequences of flaviviruses used in this manuscript. Note that WNV, and ZIKV present aromatic residues in all 3 positions. A few substitutions produce another flavivirus phenotypes in CBD sequence. 3. Chimera generation by interchange of CBD in DENV2 and YFV NS1. The complete sequence of DENV2 CBD was substituted by the ZIKV or YFV CBD sequences. The complete sequence of YFV CBD was substituted by the DENV2 or WNV CBD sequences. 4. Insertion of signal peptide of Gaussia Luc in DENV2-NS1. This insertion was introduced only in the DENV2-NS1 construct. 5. General experimental strategy with constructs. All mutations, insertions and chimera constructions were transfected in C6/36 cells and NS1 secretion to the supernatants in cells treated or not with BFA determined by ELISA.

    Journal: bioRxiv

    Article Title: The secretory fate of flavivirus NS1 in mosquito cells is influenced by the caveolin binding domain

    doi: 10.1101/2019.12.16.879031

    Figure Lengend Snippet: Schematic representation of the overall site directed mutagenesis strategy. 1. Generation of recombinant flavivirus NS1. The details for the construction of recombinants NS1 are in Materials and Methods. Each construct was used as template for site directed mutagenesis. Actin5C promoter; cherry, red fluorescent protein; eGFP, eukaryotic green fluorescent protein; neo, resistance to neomycin, kanamycin and G418, each gene separated by a T2A peptide. Yellow and blue; DENV and ZIKV NS1 CBD sequences; red; YFV NS1 incomplete CDB. 2. Substitutions of aromatic residues in CBD to Ala or Thr. CBD sequences of flaviviruses used in this manuscript. Note that WNV, and ZIKV present aromatic residues in all 3 positions. A few substitutions produce another flavivirus phenotypes in CBD sequence. 3. Chimera generation by interchange of CBD in DENV2 and YFV NS1. The complete sequence of DENV2 CBD was substituted by the ZIKV or YFV CBD sequences. The complete sequence of YFV CBD was substituted by the DENV2 or WNV CBD sequences. 4. Insertion of signal peptide of Gaussia Luc in DENV2-NS1. This insertion was introduced only in the DENV2-NS1 construct. 5. General experimental strategy with constructs. All mutations, insertions and chimera constructions were transfected in C6/36 cells and NS1 secretion to the supernatants in cells treated or not with BFA determined by ELISA.

    Article Snippet: Measurement of secreted NS1 protein The presence of flavivirus NS1 in cell supernatants was measured using a non-commercial, in-house, ELISA.

    Techniques: Mutagenesis, Recombinant, Construct, Sequencing, Transfection, Enzyme-linked Immunosorbent Assay

    Secretory phenotype of recombinant DENV (A), ZIKV (B) and YFV (C) NS1 mutated in the CBD. Left panels , schematic representation of the mutations introduced in each of the NS1 genes. Secretion mutated NS1 to Ala ( central panels ) and Thr or Phe ( right panels ) in C6/36 cells treated or not with BFA. Twenty-four hours post-transfection, C6/36 cells were treated with DMSO (control) or with 25 µM BFA and the supernatants harvested after 48 h. Levels of secreted NS1 were measured by ELISA. Data are mean of 3 independent experiments ± standard error; significant differences between controls and BFA treatment are denoted by *( p

    Journal: bioRxiv

    Article Title: The secretory fate of flavivirus NS1 in mosquito cells is influenced by the caveolin binding domain

    doi: 10.1101/2019.12.16.879031

    Figure Lengend Snippet: Secretory phenotype of recombinant DENV (A), ZIKV (B) and YFV (C) NS1 mutated in the CBD. Left panels , schematic representation of the mutations introduced in each of the NS1 genes. Secretion mutated NS1 to Ala ( central panels ) and Thr or Phe ( right panels ) in C6/36 cells treated or not with BFA. Twenty-four hours post-transfection, C6/36 cells were treated with DMSO (control) or with 25 µM BFA and the supernatants harvested after 48 h. Levels of secreted NS1 were measured by ELISA. Data are mean of 3 independent experiments ± standard error; significant differences between controls and BFA treatment are denoted by *( p

    Article Snippet: Measurement of secreted NS1 protein The presence of flavivirus NS1 in cell supernatants was measured using a non-commercial, in-house, ELISA.

    Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay

    MMP-1 expression in Bowes RGP cells promotes tumor growth and metastasis. ( a ) Bowes, Bowes-pCMV and Bowes-pCMV-MMP1 cells were injected intradermally into nude mice (10 6 cells/injection). Tumor incidence was noted (table) and tumors were measured weekly with calipers. * p

    Journal: Oncogene

    Article Title: A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

    doi: 10.1038/onc.2009.272

    Figure Lengend Snippet: MMP-1 expression in Bowes RGP cells promotes tumor growth and metastasis. ( a ) Bowes, Bowes-pCMV and Bowes-pCMV-MMP1 cells were injected intradermally into nude mice (10 6 cells/injection). Tumor incidence was noted (table) and tumors were measured weekly with calipers. * p

    Article Snippet: Mouse anti-human PAR-1 was from Beckman-Coulter (Miami, FL, USA), rabbit anti-human MMP-1 and MMP-1 neutralizing antibody were from Calbiochem, and mouse anti-FLAG from Abcam.

    Techniques: Expressing, Injection, Mouse Assay

    MMP-1 induces gene expression in VMM12 cells via PAR-1 activation. ( a ) Western blot analysis of MMP-1 and PAR-1 protein production by VMM12 cells stably transfected with scrambled control shRNA (shMAMMX), MMP-1 shRNAs (shMMP-1) and PAR-1 shRNAs (shPAR-1). PAR-1 blots were re-probed for actin, as a loading control. MMP-1 band is 54kD, PAR-1 is 61kD, actin is 43kD. ( b ) shMAMMX, shMMP-1 and shPAR-1 cells were treated with media conditioned by the same cell line for 24hr, with MMPs activated as described. Gene expression was measured by realtime RT-PCR. * p ≤0.002, compared to shMMP-1 gene expression, ** p ≤0.025, compared to shMAMMX gene expression. ( c ) shMMP-1 cells were treated with either DMSO (control), 5nM activated MMP-1 or 5nM MMP-1+50nM SCH79797. After 24hr, cells were harvested and gene expression measured by realtime-RT PCR. # p ≤0.003 compared to shMMP-1 control, ## p ≤0.005, compared to treatment with 5nM MMP-1. For all, data were normalized to GAPDH, and were analyzed by the 2 ΔΔC(t) method, and are representative of 3 experiments.

    Journal: Oncogene

    Article Title: A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

    doi: 10.1038/onc.2009.272

    Figure Lengend Snippet: MMP-1 induces gene expression in VMM12 cells via PAR-1 activation. ( a ) Western blot analysis of MMP-1 and PAR-1 protein production by VMM12 cells stably transfected with scrambled control shRNA (shMAMMX), MMP-1 shRNAs (shMMP-1) and PAR-1 shRNAs (shPAR-1). PAR-1 blots were re-probed for actin, as a loading control. MMP-1 band is 54kD, PAR-1 is 61kD, actin is 43kD. ( b ) shMAMMX, shMMP-1 and shPAR-1 cells were treated with media conditioned by the same cell line for 24hr, with MMPs activated as described. Gene expression was measured by realtime RT-PCR. * p ≤0.002, compared to shMMP-1 gene expression, ** p ≤0.025, compared to shMAMMX gene expression. ( c ) shMMP-1 cells were treated with either DMSO (control), 5nM activated MMP-1 or 5nM MMP-1+50nM SCH79797. After 24hr, cells were harvested and gene expression measured by realtime-RT PCR. # p ≤0.003 compared to shMMP-1 control, ## p ≤0.005, compared to treatment with 5nM MMP-1. For all, data were normalized to GAPDH, and were analyzed by the 2 ΔΔC(t) method, and are representative of 3 experiments.

    Article Snippet: Mouse anti-human PAR-1 was from Beckman-Coulter (Miami, FL, USA), rabbit anti-human MMP-1 and MMP-1 neutralizing antibody were from Calbiochem, and mouse anti-FLAG from Abcam.

    Techniques: Expressing, Activation Assay, Western Blot, Stable Transfection, Transfection, shRNA, Reverse Transcription Polymerase Chain Reaction

    Both the collagenase and PAR-1 activating functions of MMP-1 are required for melanoma cell invasion. ( a ) VMM12 shRNA lines were used in a type I collagen degradation assay. Cells were embedded in type I collagen, and after 48hr, the media released from the collagen gel were weighed to determine the amount of collagen that had been degraded. # p

    Journal: Oncogene

    Article Title: A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

    doi: 10.1038/onc.2009.272

    Figure Lengend Snippet: Both the collagenase and PAR-1 activating functions of MMP-1 are required for melanoma cell invasion. ( a ) VMM12 shRNA lines were used in a type I collagen degradation assay. Cells were embedded in type I collagen, and after 48hr, the media released from the collagen gel were weighed to determine the amount of collagen that had been degraded. # p

    Article Snippet: Mouse anti-human PAR-1 was from Beckman-Coulter (Miami, FL, USA), rabbit anti-human MMP-1 and MMP-1 neutralizing antibody were from Calbiochem, and mouse anti-FLAG from Abcam.

    Techniques: shRNA, Degradation Assay

    MMP-1 expression in Bowes RGP cells induces some aspects of the VGP phenotype in vitro , via PAR-1 activation. ( a ) Bowes cells were stably transfected with pCMV (empty vector control) or pCMV-MMP1. MMP-1 and PAR-1 protein levels were measured by western blot. PAR-1 blots were re-probed for actin, as a loading control. MMP-1 band is 54kD, PAR-1 is 61kD, actin is 43kD. ( b ) Bowe-pCMV and Bowes-pCMV-MMP1 cells were serum-starved for 2hr, then treated for 15′ with media from the same cell line, with MMPs activated as described. Media were treated with either DMSO (-), 5μM MMP inhibitor II, or cells were pre-treated with 50nM SCH79797, as indicated. The phosphorylation status of MEK1/2 and p38 were examined by western blot of the cell lysates. Blots were re-probed with antibodies against the corresponding total protein. MEK1/2 band size is 44kD, p38 is 38kD. ( c ) Realtime RT-PCR was used to measure the expression of selected genes in cells treated with media conditioned by the same cell line, with MMPs activated. Cells were treated with either DMSO or 50nM SCH79797. Data are normalized to GAPDH expression and were analyzed using the 2 -ΔΔC(t) method. * p ≤0.002, compared to pCMV gene expression, ** p ≤0.015, compared to Bowes-MMP1 gene expression. ( d ) Cells were plated in media conditioned by the same cell line, with MMPs activated, and viable cells were counted after 48, 96, and 144hr. Cells were treated with either DMSO or 50μM SCH79797. # p

    Journal: Oncogene

    Article Title: A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

    doi: 10.1038/onc.2009.272

    Figure Lengend Snippet: MMP-1 expression in Bowes RGP cells induces some aspects of the VGP phenotype in vitro , via PAR-1 activation. ( a ) Bowes cells were stably transfected with pCMV (empty vector control) or pCMV-MMP1. MMP-1 and PAR-1 protein levels were measured by western blot. PAR-1 blots were re-probed for actin, as a loading control. MMP-1 band is 54kD, PAR-1 is 61kD, actin is 43kD. ( b ) Bowe-pCMV and Bowes-pCMV-MMP1 cells were serum-starved for 2hr, then treated for 15′ with media from the same cell line, with MMPs activated as described. Media were treated with either DMSO (-), 5μM MMP inhibitor II, or cells were pre-treated with 50nM SCH79797, as indicated. The phosphorylation status of MEK1/2 and p38 were examined by western blot of the cell lysates. Blots were re-probed with antibodies against the corresponding total protein. MEK1/2 band size is 44kD, p38 is 38kD. ( c ) Realtime RT-PCR was used to measure the expression of selected genes in cells treated with media conditioned by the same cell line, with MMPs activated. Cells were treated with either DMSO or 50nM SCH79797. Data are normalized to GAPDH expression and were analyzed using the 2 -ΔΔC(t) method. * p ≤0.002, compared to pCMV gene expression, ** p ≤0.015, compared to Bowes-MMP1 gene expression. ( d ) Cells were plated in media conditioned by the same cell line, with MMPs activated, and viable cells were counted after 48, 96, and 144hr. Cells were treated with either DMSO or 50μM SCH79797. # p

    Article Snippet: Mouse anti-human PAR-1 was from Beckman-Coulter (Miami, FL, USA), rabbit anti-human MMP-1 and MMP-1 neutralizing antibody were from Calbiochem, and mouse anti-FLAG from Abcam.

    Techniques: Expressing, In Vitro, Activation Assay, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Factors in the tumor microenvironment may induce MMP-1 expression in Bowes RGP melanoma cells, and MMP-1 strongly induces MMP-1 expression via PAR-1. ( a ) Bowes cells were treated for 24hr in serum-free media with 5nM thrombin, 25ng/mL bFGF, 10ng/mL VEGF. MMP-1 expression was measured by realtime-RT PCR. Data were normalized to GAPDH, and analyzed by the 2 ΔΔC(t) method. * p =0.015, ** p =0.002, *** p =0.042, compared to control. The corresponding western blot is also shown, with an exposure time of 5 minutes. The MMP-1 band size is 54kD. ( b ) Bowes cells were treated with DMSO, 5nM MMP-1 or 5nM MMP-1+50nM SCH79797. After 24hr, MMP-1 expression was measured by realtime-RT PCR. Data were normalized to GAPDH expression and analyzed using the 2 (-ΔΔCt) method. † p

    Journal: Oncogene

    Article Title: A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

    doi: 10.1038/onc.2009.272

    Figure Lengend Snippet: Factors in the tumor microenvironment may induce MMP-1 expression in Bowes RGP melanoma cells, and MMP-1 strongly induces MMP-1 expression via PAR-1. ( a ) Bowes cells were treated for 24hr in serum-free media with 5nM thrombin, 25ng/mL bFGF, 10ng/mL VEGF. MMP-1 expression was measured by realtime-RT PCR. Data were normalized to GAPDH, and analyzed by the 2 ΔΔC(t) method. * p =0.015, ** p =0.002, *** p =0.042, compared to control. The corresponding western blot is also shown, with an exposure time of 5 minutes. The MMP-1 band size is 54kD. ( b ) Bowes cells were treated with DMSO, 5nM MMP-1 or 5nM MMP-1+50nM SCH79797. After 24hr, MMP-1 expression was measured by realtime-RT PCR. Data were normalized to GAPDH expression and analyzed using the 2 (-ΔΔCt) method. † p

    Article Snippet: Mouse anti-human PAR-1 was from Beckman-Coulter (Miami, FL, USA), rabbit anti-human MMP-1 and MMP-1 neutralizing antibody were from Calbiochem, and mouse anti-FLAG from Abcam.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    PAR-1 cleavage by MMP-1 occurs in VMM12 VGP melanoma cells. ( a ) Western blot analysis of MMP-1 protein production by normal melanocytes, Bowes RGP melanoma cells, and VMM12 VGP melanoma cells, and analysis PAR-1 protein expression by normal endothelial cells, Bowes and VMM12 cells. PAR-1 blots were re-probed for actin, as a loading control. MMP-1 band is 54kD, PAR-1 is 61kD, actin is 43kD. ( b ) VMM12 cells were transfected with AP-PAR1, and treated with media conditioned for 24hr by either Bowes or VMM12 cells. The amount of alkaline phosphatase in the media, due to PAR-1 cleavage, was measured after 1hr. ( c ) VMM12 conditioned media (CM) were treated with either DMSO, 0.05U/mL hirudin (thrombin inhibitor), 5μM MMP inhibitor II, which blocks activity of MMP-1,-3,-7,-9 or 5μM MMP inhibitor V, which blocks MMP-2,-3,-8,-9,-12,-13 activity. MMP-1 neutralizing antibody or anti-FLAG (IgG control) were added at the indicated concentration to VMM12 CM. Media were used to treat AP-PAR1 transfected VMM12 cells for 1hr. Alkaline phophatase activity was measured to quantify PAR-1 cleavage. * p =0.02 and ** p

    Journal: Oncogene

    Article Title: A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

    doi: 10.1038/onc.2009.272

    Figure Lengend Snippet: PAR-1 cleavage by MMP-1 occurs in VMM12 VGP melanoma cells. ( a ) Western blot analysis of MMP-1 protein production by normal melanocytes, Bowes RGP melanoma cells, and VMM12 VGP melanoma cells, and analysis PAR-1 protein expression by normal endothelial cells, Bowes and VMM12 cells. PAR-1 blots were re-probed for actin, as a loading control. MMP-1 band is 54kD, PAR-1 is 61kD, actin is 43kD. ( b ) VMM12 cells were transfected with AP-PAR1, and treated with media conditioned for 24hr by either Bowes or VMM12 cells. The amount of alkaline phosphatase in the media, due to PAR-1 cleavage, was measured after 1hr. ( c ) VMM12 conditioned media (CM) were treated with either DMSO, 0.05U/mL hirudin (thrombin inhibitor), 5μM MMP inhibitor II, which blocks activity of MMP-1,-3,-7,-9 or 5μM MMP inhibitor V, which blocks MMP-2,-3,-8,-9,-12,-13 activity. MMP-1 neutralizing antibody or anti-FLAG (IgG control) were added at the indicated concentration to VMM12 CM. Media were used to treat AP-PAR1 transfected VMM12 cells for 1hr. Alkaline phophatase activity was measured to quantify PAR-1 cleavage. * p =0.02 and ** p

    Article Snippet: Mouse anti-human PAR-1 was from Beckman-Coulter (Miami, FL, USA), rabbit anti-human MMP-1 and MMP-1 neutralizing antibody were from Calbiochem, and mouse anti-FLAG from Abcam.

    Techniques: Western Blot, Expressing, Transfection, Activity Assay, Concentration Assay