protease inhibitor mix  (Millipore)

 
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    Name:
    Protease Inhibitor Cocktail
    Description:

    Catalog Number:
    p8340
    Price:
    None
    Applications:
    Protease Inhibitor Cocktail has been used-. as a buffer component during sonication of GFP (green fluorescent protein)-huntingtin-transfected HEK 293 cells in GST (glutathione S-transferase) pull down assay. as a component of lysis buffer. as a component of radioimmunoprecipitation assay buffer (RIPA)
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    Structured Review

    Millipore protease inhibitor mix

    https://www.bioz.com/result/protease inhibitor mix/product/Millipore
    Average 99 stars, based on 278 article reviews
    Price from $9.99 to $1999.99
    protease inhibitor mix - by Bioz Stars, 2021-02
    99/100 stars

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    Protease Inhibitor:

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells
    Article Snippet: .. Phenylmethylsulfonylfuoride (PMSF) (Euroclone, Devon, UK), pepstatin A (Boehringer, Mannheim, DE), GM6001 inhibitor (Biomol Research Lab Inc.), protease inhibitor cocktail set III (AEBSF 100mM; aprotinin 80μM; bestatin 5mM; E-64 1,5mM; leupeptin 2mM; pepstatin A 1mM) (Calbiochem, La Jolla, CA) were used for specific protease class inhibition experiments. .. Ox-LDL preparation, labelling and fluorescence analysis Human LDL was prepared from fresh healthy normolipidemic plasma of volunteers by ultracentrifugation [ ] and in vitro oxidized as described (Matarazzo et al., 2012).

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Article Title: Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace
    Article Snippet: .. For protein lysate samples, one of the 0.5 mL BAL aliquots was treated with 10 μL protease inhibitor cocktail set III, EDTA-free (Calbiochem, MA) and 20 μl of 10% Triton X-100 (Sigma, MA) and mixed by inversion for 20 minutes at 4 °C. .. Protein lysate samples were used for mouse neutrophil elastase/ELA2 and MPO ELISA (R & D Systems, MN) per manufacturers protocol.

    Article Title: Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue ▿Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue ▿ †Nasal Immunity to Staphylococcal Toxic Shock Is Controlled by the Nasopharynx-Associated Lymphoid Tissue ▿ † ‡
    Article Snippet: .. The collected rinse (5 to 8 μl) was deposited in a fresh centrifuge tube containing 10 μl PBS with 2× protease inhibitor cocktail set III (Calbiochem, San Diego, CA). .. Nasal lavage was collected from euthanized (CO2 asphyxiation, followed by cervical dislocation) mice by carefully introducing 20 to 30 μl PBS with a pipette through one nostril.

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Cell Culture:

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Immunoprecipitation:

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Incubation:

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Inhibition:

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells
    Article Snippet: .. Phenylmethylsulfonylfuoride (PMSF) (Euroclone, Devon, UK), pepstatin A (Boehringer, Mannheim, DE), GM6001 inhibitor (Biomol Research Lab Inc.), protease inhibitor cocktail set III (AEBSF 100mM; aprotinin 80μM; bestatin 5mM; E-64 1,5mM; leupeptin 2mM; pepstatin A 1mM) (Calbiochem, La Jolla, CA) were used for specific protease class inhibition experiments. .. Ox-LDL preparation, labelling and fluorescence analysis Human LDL was prepared from fresh healthy normolipidemic plasma of volunteers by ultracentrifugation [ ] and in vitro oxidized as described (Matarazzo et al., 2012).

    Fractionation:

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Lysis:

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses
    Article Snippet: .. Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation. ..

    Article Title: The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion
    Article Snippet: .. Cells were resuspended in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM EDTA, 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitor cocktail III [Calbiochem]) and incubated for 10 min on ice prior to addition of an equal amount of ice-cold water. .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce).

    Modification:

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
    Article Snippet: .. Co-immunoprecipitation Uninfected or E. chaffeensis-infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805). .. Normal rabbit (Santa Cruz Biotechnology, sc-2027) or mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as a negative control for immunoprecipitation.

    Western Blot:

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

    Recombinant:

    Article Title: Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins
    Article Snippet: .. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail). .. Production of recombinant schistosome membrane proteins in E . coli DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the E . coli thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags.

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  • 99
    Millipore z vad fmk
    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM <t>z-VAD-fmk</t> or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.
    Z Vad Fmk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/z vad fmk/product/Millipore
    Average 99 stars, based on 187 article reviews
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    99
    Millipore pepstatin a
    Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP  in vitro . A , cleavage of 5 μ m  Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 m m  Ca-DTPA, 50 μ m  TPEN, 18 μ m  SB3CT (  43 ), 50 n m  GM6001 (also tested at 1 μ m  and no inhibition; data not shown), 0.26 μ m  prinomastat, 150 n m  aprotinin, 10 μ m  E-64, 100 μ m  leupeptin, 1 μ m  pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity.  B , cleavage of RLQLKL ACPP for 2 h with 50 n m  MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen ( supplemental Fig. 4 ). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in  supplemental Fig. 4 .
    Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 396 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pepstatin a/product/Millipore
    Average 99 stars, based on 396 article reviews
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    99
    Millipore complete protease inhibitor
    DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in <t>complete</t> medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.
    Complete Protease Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete protease inhibitor/product/Millipore
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    89
    Millipore drg tissues
    Double immunostaining (IS) of <t>Piezo2</t> (PZ2) with a selection of neuronal and glial markers. Representative montage images of <t>DRG</t> sections show Piezo2-IR (red) co-stained with a selection of DRG neuronal markers (green), including Tubb3 ( A ), IB4 ( B ), CGRP ( C ), NF200 ( D ), TrkB ( E ), and NKA1 α ( F ) with the squared region shown at high magnification ( F1 ). The panels in the right-side of A - E calculate the percentage of Piezo2-neurons overlaid to Tubb3-neurons ( A 1), as well as neurons positive for IB4 ( B1 ), CGRP ( C1 ), NF200 ( D1 ), and TrkB ( E1 ) overlaid to Piezo2-neurons. The numbers are the counted Piezo2-IR neurons (red) and marker-labeled neurons (green). ICA analyzes colocalization between Piezo2 and NKA1 α by an ImageJ 1.46r software plugin colocalization analysis module ( G ). Scatter plots for the region demarcated by the white dashed line in F1 panel show data clustered along both positive and negative axes for both Piezo2 and NKA1 α . “A” is the intensity of Piezo2 while “a” is the average of these values, and “B” is the intensity of NKA1a while “b” is the average of these values. For this region, the ICQ value is 0.127 (P sign test
    Drg Tissues, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Article Snippet: JC-1, antibiotic, AlexaFluor 488 and 568 conjugated antibodies, Lipofectamine 2000 were purchased from Invitrogen; z-VAD-fmk, protease inhibitor cocktail and GAPDH antibody were from Millipore.

    Techniques: Transfection, Activity Assay, Staining, Flow Cytometry, Cytometry, Sulforhodamine B Assay

    Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP  in vitro . A , cleavage of 5 μ m  Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 m m  Ca-DTPA, 50 μ m  TPEN, 18 μ m  SB3CT (  43 ), 50 n m  GM6001 (also tested at 1 μ m  and no inhibition; data not shown), 0.26 μ m  prinomastat, 150 n m  aprotinin, 10 μ m  E-64, 100 μ m  leupeptin, 1 μ m  pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity.  B , cleavage of RLQLKL ACPP for 2 h with 50 n m  MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen ( supplemental Fig. 4 ). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in  supplemental Fig. 4 .

    Journal: The Journal of Biological Chemistry

    Article Title: Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides *

    doi: 10.1074/jbc.M110.138297

    Figure Lengend Snippet: Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP in vitro . A , cleavage of 5 μ m Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 m m Ca-DTPA, 50 μ m TPEN, 18 μ m SB3CT ( 43 ), 50 n m GM6001 (also tested at 1 μ m and no inhibition; data not shown), 0.26 μ m prinomastat, 150 n m aprotinin, 10 μ m E-64, 100 μ m leupeptin, 1 μ m pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity. B , cleavage of RLQLKL ACPP for 2 h with 50 n m MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen ( supplemental Fig. 4 ). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in supplemental Fig. 4 .

    Article Snippet: Tumors were excised and homogenized in PBS in the presence of protease inhibitor containing 100 μm 4-(2-aminoethyl)-benzenesulfonyl fluoride-HCl, 80 nm aprotinin, 5 μm bestatin, 1.5 μm E-64, 2 μm leupeptin hemisulfate, and 1 μm pepstatin A (EMD Biosciences mixture III).

    Techniques: In Vitro, Labeling, Inhibition, Activity Assay, Software

    DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in complete medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in complete medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Cell Cycle Assay, Staining, Incubation, Neutral Comet Assay, Software, Cell Culture, Crystal Violet Assay, Standard Deviation

    DAG-induced DNA damage activates HR pathway. a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: DAG-induced DNA damage activates HR pathway. a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Incubation, Western Blot, Transfection, Negative Control, Crystal Violet Assay, Standard Deviation, Expressing

    Cytotoxic effect of DAG in GBM and PCa cell lines. a M059K and PC-3 cells were treated with or without 10 μM DAG in complete medium for 72 h. The representative bright-field images were shown and the scale bar represents 100 μm. b , c Two GBM (M059K and M059J) and four PCa (PC-3, LNCaP, DU-145, and PC-3-DR) cell lines were treated with different concentrations of DAG (0, 50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 72 h in 96-well culture plates. Following the treatment, crystal violet assay was performed and the IC 50 values of DAG for each cell line were calculated by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The data on the curve are presented as mean ± standard deviation. Three individual experiments were performed for each cell line.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: Cytotoxic effect of DAG in GBM and PCa cell lines. a M059K and PC-3 cells were treated with or without 10 μM DAG in complete medium for 72 h. The representative bright-field images were shown and the scale bar represents 100 μm. b , c Two GBM (M059K and M059J) and four PCa (PC-3, LNCaP, DU-145, and PC-3-DR) cell lines were treated with different concentrations of DAG (0, 50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 72 h in 96-well culture plates. Following the treatment, crystal violet assay was performed and the IC 50 values of DAG for each cell line were calculated by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The data on the curve are presented as mean ± standard deviation. Three individual experiments were performed for each cell line.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Crystal Violet Assay, Standard Deviation

    Double immunostaining (IS) of Piezo2 (PZ2) with a selection of neuronal and glial markers. Representative montage images of DRG sections show Piezo2-IR (red) co-stained with a selection of DRG neuronal markers (green), including Tubb3 ( A ), IB4 ( B ), CGRP ( C ), NF200 ( D ), TrkB ( E ), and NKA1 α ( F ) with the squared region shown at high magnification ( F1 ). The panels in the right-side of A - E calculate the percentage of Piezo2-neurons overlaid to Tubb3-neurons ( A 1), as well as neurons positive for IB4 ( B1 ), CGRP ( C1 ), NF200 ( D1 ), and TrkB ( E1 ) overlaid to Piezo2-neurons. The numbers are the counted Piezo2-IR neurons (red) and marker-labeled neurons (green). ICA analyzes colocalization between Piezo2 and NKA1 α by an ImageJ 1.46r software plugin colocalization analysis module ( G ). Scatter plots for the region demarcated by the white dashed line in F1 panel show data clustered along both positive and negative axes for both Piezo2 and NKA1 α . “A” is the intensity of Piezo2 while “a” is the average of these values, and “B” is the intensity of NKA1a while “b” is the average of these values. For this region, the ICQ value is 0.127 (P sign test

    Journal: bioRxiv

    Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

    doi: 10.1101/2021.01.20.427483

    Figure Lengend Snippet: Double immunostaining (IS) of Piezo2 (PZ2) with a selection of neuronal and glial markers. Representative montage images of DRG sections show Piezo2-IR (red) co-stained with a selection of DRG neuronal markers (green), including Tubb3 ( A ), IB4 ( B ), CGRP ( C ), NF200 ( D ), TrkB ( E ), and NKA1 α ( F ) with the squared region shown at high magnification ( F1 ). The panels in the right-side of A - E calculate the percentage of Piezo2-neurons overlaid to Tubb3-neurons ( A 1), as well as neurons positive for IB4 ( B1 ), CGRP ( C1 ), NF200 ( D1 ), and TrkB ( E1 ) overlaid to Piezo2-neurons. The numbers are the counted Piezo2-IR neurons (red) and marker-labeled neurons (green). ICA analyzes colocalization between Piezo2 and NKA1 α by an ImageJ 1.46r software plugin colocalization analysis module ( G ). Scatter plots for the region demarcated by the white dashed line in F1 panel show data clustered along both positive and negative axes for both Piezo2 and NKA1 α . “A” is the intensity of Piezo2 while “a” is the average of these values, and “B” is the intensity of NKA1a while “b” is the average of these values. For this region, the ICQ value is 0.127 (P sign test

    Article Snippet: The collected cell pellets and pooled L4/L5 DRG from CFA and control rats were extracted using 1x RIPA ice-cold buffer (20 mm Tris-HCl pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, with 0.1% Triton X100 and protease inhibitor cocktail) and rotated at 4°C for 1 h before the supernatant was extracted by centrifugation at 12,000 g at 4°C for 5 min. To examine the subcellular localization of Piezo2, DRG tissues were homogenized and then fractionated to obtain plasma membrane and cytosolic fractions, using the ProteoExtract Subcellular Proteome Extraction Kit (Millipore, Billerica, MA), according to the manufacturer’s instructions.

    Techniques: Double Immunostaining, Selection, Staining, Marker, Labeling, Software

    Specificity of Piezo2 (PZ2) antibody in detection of Piezo2 expression. Several bands around 310-150KDa are detected by immunoblot (IB) in the lysates of neuronal cell lines, which are eliminated by preincubation with blocking peptide (BP) ( A ). In DRG lysate, the antibody detects several bands at approximately 310, 240, 180, and 130 KDa and marked as putative “isoforms (iso)” 1-4, respectively, of the target protein since they are eliminated by preincubation with BP ( B ). Piezo2 IB in the cytosolic and membrane fractions prepared from pooled L4/L5 DRG show that the putative Piezo2 iso2 (∼240KDa) and iso3 (∼180KDa) are more enriched in membranes while the canonical Piezo2 (KDa ∼310) and putative iso4 (KDa ∼130) are more localized in cytosol ( C ). Representative images of double immunostaining (IS) of Piezo2 (red) and Tubb3 (green) on DRG sections reveal pan-neuronal Piezo2-IR ( D ) and preincubation with BP eliminated the staining ( D1 ). Representative images of double-IS of Piezo2 (red) and Tubb3 (green) on trigeminal ganglia (TG) sections reveal pan-neuronal Piezo2-IR ( E ). Scale bars: 100 μm for all. Expression plasmid map ( F ) in which U6 and H1 promoters transcribe convergent apposing Piezo2-shRNA expression. The Piezo2-shRNA cassette (sequences shown on the top of plasmid map) is cloned into Mlul site (pointed by arrowhead) of plasmid and named pU6/H1-Piezo2shRNA-CMV-EGFP (Piezo2-shRNA). Transfection of Piezo2-shRNA into N2A cells yielded 70-80% transfection rate (n=4) ( G ); Piezo2 protein in N2A lysates analyzed by IB using ProSci Piezo2 antibody: Lane 1, mock transfection; lane 2, transfection with scrambled RNA; lane 3-4, duplicate transfection using Piezo2-shRNA ( H ); Densitometry of IBs of fold change in Piezo2 protein (left) and qPCR (right) show fold change in Piezo2 mRNA (**p

    Journal: bioRxiv

    Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

    doi: 10.1101/2021.01.20.427483

    Figure Lengend Snippet: Specificity of Piezo2 (PZ2) antibody in detection of Piezo2 expression. Several bands around 310-150KDa are detected by immunoblot (IB) in the lysates of neuronal cell lines, which are eliminated by preincubation with blocking peptide (BP) ( A ). In DRG lysate, the antibody detects several bands at approximately 310, 240, 180, and 130 KDa and marked as putative “isoforms (iso)” 1-4, respectively, of the target protein since they are eliminated by preincubation with BP ( B ). Piezo2 IB in the cytosolic and membrane fractions prepared from pooled L4/L5 DRG show that the putative Piezo2 iso2 (∼240KDa) and iso3 (∼180KDa) are more enriched in membranes while the canonical Piezo2 (KDa ∼310) and putative iso4 (KDa ∼130) are more localized in cytosol ( C ). Representative images of double immunostaining (IS) of Piezo2 (red) and Tubb3 (green) on DRG sections reveal pan-neuronal Piezo2-IR ( D ) and preincubation with BP eliminated the staining ( D1 ). Representative images of double-IS of Piezo2 (red) and Tubb3 (green) on trigeminal ganglia (TG) sections reveal pan-neuronal Piezo2-IR ( E ). Scale bars: 100 μm for all. Expression plasmid map ( F ) in which U6 and H1 promoters transcribe convergent apposing Piezo2-shRNA expression. The Piezo2-shRNA cassette (sequences shown on the top of plasmid map) is cloned into Mlul site (pointed by arrowhead) of plasmid and named pU6/H1-Piezo2shRNA-CMV-EGFP (Piezo2-shRNA). Transfection of Piezo2-shRNA into N2A cells yielded 70-80% transfection rate (n=4) ( G ); Piezo2 protein in N2A lysates analyzed by IB using ProSci Piezo2 antibody: Lane 1, mock transfection; lane 2, transfection with scrambled RNA; lane 3-4, duplicate transfection using Piezo2-shRNA ( H ); Densitometry of IBs of fold change in Piezo2 protein (left) and qPCR (right) show fold change in Piezo2 mRNA (**p

    Article Snippet: The collected cell pellets and pooled L4/L5 DRG from CFA and control rats were extracted using 1x RIPA ice-cold buffer (20 mm Tris-HCl pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, with 0.1% Triton X100 and protease inhibitor cocktail) and rotated at 4°C for 1 h before the supernatant was extracted by centrifugation at 12,000 g at 4°C for 5 min. To examine the subcellular localization of Piezo2, DRG tissues were homogenized and then fractionated to obtain plasma membrane and cytosolic fractions, using the ProteoExtract Subcellular Proteome Extraction Kit (Millipore, Billerica, MA), according to the manufacturer’s instructions.

    Techniques: Expressing, Blocking Assay, Double Immunostaining, Staining, Plasmid Preparation, shRNA, Clone Assay, Transfection, Real-time Polymerase Chain Reaction

    Validation of Piezos (2 and 1) expression by RT-PCR RT-PCR shows amplification of Piezo1 and Piezo2 transcripts in rat 50B11 cells ( A ), rat DRG ( B ), rat primary cultured SGCs at day of in vitro 4 (DIV, C ), and rat primary cultured Schwann cells at DIV5 ( D ) by two different primer pairs specific for amplification of rat Piezo1 and Piezo2 transcripts, as well as detection of Piezo1 and Piezo2 transcripts by two different primer pairs specific for human Piezo1 and 2 in human primary cultured melanocytes ( E ). Lane 1: ladder, lane 2-3: Piezo1 amplified by two piezo1 primer pairs ( A-D , rat; E , human), lane 3-4: Piezo2 amplified by two piezo2 primer pairs ( A-D , rat; E , human), lane 7: Gapdh, and lane 8: negative control.

    Journal: bioRxiv

    Article Title: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

    doi: 10.1101/2021.01.20.427483

    Figure Lengend Snippet: Validation of Piezos (2 and 1) expression by RT-PCR RT-PCR shows amplification of Piezo1 and Piezo2 transcripts in rat 50B11 cells ( A ), rat DRG ( B ), rat primary cultured SGCs at day of in vitro 4 (DIV, C ), and rat primary cultured Schwann cells at DIV5 ( D ) by two different primer pairs specific for amplification of rat Piezo1 and Piezo2 transcripts, as well as detection of Piezo1 and Piezo2 transcripts by two different primer pairs specific for human Piezo1 and 2 in human primary cultured melanocytes ( E ). Lane 1: ladder, lane 2-3: Piezo1 amplified by two piezo1 primer pairs ( A-D , rat; E , human), lane 3-4: Piezo2 amplified by two piezo2 primer pairs ( A-D , rat; E , human), lane 7: Gapdh, and lane 8: negative control.

    Article Snippet: The collected cell pellets and pooled L4/L5 DRG from CFA and control rats were extracted using 1x RIPA ice-cold buffer (20 mm Tris-HCl pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, with 0.1% Triton X100 and protease inhibitor cocktail) and rotated at 4°C for 1 h before the supernatant was extracted by centrifugation at 12,000 g at 4°C for 5 min. To examine the subcellular localization of Piezo2, DRG tissues were homogenized and then fractionated to obtain plasma membrane and cytosolic fractions, using the ProteoExtract Subcellular Proteome Extraction Kit (Millipore, Billerica, MA), according to the manufacturer’s instructions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Cell Culture, In Vitro, Negative Control