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  • 99
    Thermo Fisher ne per nuclear
    Localisation of galectin-3 and hnRNPA2B1 in cell nuclei. a <t>HeLa</t> cells were fixed and stained by immunofluorescence with anti-galectin-3/ Alexa Fluor 546 together with anti-hnRNPA2B1/ Alexa Fluor 647. Punctate structures positive for hnRNPA2B1 and endogenous galectin-3 are indicated by arrows. Nuclear staining (DAPI) is depicted in blue, scale bars: 10 μm. b Interaction between galectin-3 and hnRNPA2B1 was assessed by in situ PLA. HeLa cells were fixed and incubated with antibodies directed against galectin-3 and hnRNPA2B1. In the negative control HeLa cells were incubated in the absence of primary antibodies. HeLa cells were incubated with primary antibodies against hnRNPA2B1 and Sc35 as positive control. Interactions in a proximity up to 40 nm appear as fluorescent dots. Nuclei were stained with DAPI. For a better comparison, nuclei are depicted in blue, cytoplasmic PLA-signals in magenta and nuclear PLA-signals in dark blue in the merged images. Scale bars; 10 μm. c The amount of PLA-spots <t>per</t> nucleus was quantified. Bar graphs indicate the average relative number of PLA-signals per nucleus +/− SD, n = 3 (** p = 0.01)
    Ne Per Nuclear, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore petri dishes
    Localisation of galectin-3 and hnRNPA2B1 in cell nuclei. a <t>HeLa</t> cells were fixed and stained by immunofluorescence with anti-galectin-3/ Alexa Fluor 546 together with anti-hnRNPA2B1/ Alexa Fluor 647. Punctate structures positive for hnRNPA2B1 and endogenous galectin-3 are indicated by arrows. Nuclear staining (DAPI) is depicted in blue, scale bars: 10 μm. b Interaction between galectin-3 and hnRNPA2B1 was assessed by in situ PLA. HeLa cells were fixed and incubated with antibodies directed against galectin-3 and hnRNPA2B1. In the negative control HeLa cells were incubated in the absence of primary antibodies. HeLa cells were incubated with primary antibodies against hnRNPA2B1 and Sc35 as positive control. Interactions in a proximity up to 40 nm appear as fluorescent dots. Nuclei were stained with DAPI. For a better comparison, nuclei are depicted in blue, cytoplasmic PLA-signals in magenta and nuclear PLA-signals in dark blue in the merged images. Scale bars; 10 μm. c The amount of PLA-spots <t>per</t> nucleus was quantified. Bar graphs indicate the average relative number of PLA-signals per nucleus +/− SD, n = 3 (** p = 0.01)
    Petri Dishes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore percoll
    Activities of creatinase expressed in spore wall deficient mutants, osw2Δ, dit1Δ , and chs3Δ . (A) 50 µg of lysates from wild-type (wt), osw2Δ, dit1Δ , or chs3Δ spores expressing creatinase–HA were subjected to western blot analysis using an anti–HA antibody to detect the HA fusion (arrow). Wild-type spores harboring the empty vector was used as a control. (B, C) Creatinase activity was measured for 28 mg of indicated wet spores (B) or 5 mg of freeze-dried spores (C) expressing creatinase–HA. Spore samples were purified by <t>percoll</t> gradient centrifugation before measuring weight or freeze-drying. Wild-type spores harboring the empty vector were used as a control. Data presented are the mean ± SE of 3 independent samples. * P
    Percoll, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7671 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ampicillin
    Activities of creatinase expressed in spore wall deficient mutants, osw2Δ, dit1Δ , and chs3Δ . (A) 50 µg of lysates from wild-type (wt), osw2Δ, dit1Δ , or chs3Δ spores expressing creatinase–HA were subjected to western blot analysis using an anti–HA antibody to detect the HA fusion (arrow). Wild-type spores harboring the empty vector was used as a control. (B, C) Creatinase activity was measured for 28 mg of indicated wet spores (B) or 5 mg of freeze-dried spores (C) expressing creatinase–HA. Spore samples were purified by <t>percoll</t> gradient centrifugation before measuring weight or freeze-drying. Wild-type spores harboring the empty vector were used as a control. Data presented are the mean ± SE of 3 independent samples. * P
    Ampicillin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fbs
    Activities of creatinase expressed in spore wall deficient mutants, osw2Δ, dit1Δ , and chs3Δ . (A) 50 µg of lysates from wild-type (wt), osw2Δ, dit1Δ , or chs3Δ spores expressing creatinase–HA were subjected to western blot analysis using an anti–HA antibody to detect the HA fusion (arrow). Wild-type spores harboring the empty vector was used as a control. (B, C) Creatinase activity was measured for 28 mg of indicated wet spores (B) or 5 mg of freeze-dried spores (C) expressing creatinase–HA. Spore samples were purified by <t>percoll</t> gradient centrifugation before measuring weight or freeze-drying. Wild-type spores harboring the empty vector were used as a control. Data presented are the mean ± SE of 3 independent samples. * P
    Fbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore edta
    The bulk of milk microRNA pellets at low ultracentrifugation speeds and microRNAs distribution do not correspond to EV-associated proteins profiles. (a) Commercial milk preparation (100 mL) was subjected to successive differential ultracentrifugation steps at 12,000 g , 35,000 g , 70,000 g and 100,000 g for 1 h each at 4°C, and each pellet was kept and suspended in 1 mL <t>PBS</t> containing <t>EDTA,</t> as detailed in the Material and methods section. (b) Each sample (250 µL) was mixed with 750 µL of TRIzol-LS reagent for total RNA isolation and subsequent RT-qPCR detection of microRNAs bta-miR-223, bta-miR-125b, bta-miR-148a, bta-miR-29b, bta-miR-151-3p and bta-miR-2478. The experiment was performed three times with three different milk samples, and each quantification level in each pellet was reported on the sum of all the pellet (total) and expressed as a percentage of the total (mean ± SD; n = 3 or 6). The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post hoc comparison of the means with Tukey’s correction with p
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore triton x 100
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore penicillin streptomycin
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Penicillin Streptomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore protease inhibitor cocktail
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Protease Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 134786 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore centrifuge tubes
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Centrifuge Tubes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore orbital shaker
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Orbital Shaker, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore kanamycin
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Kanamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7529 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher topo ta cloning kit
    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pbs
    Effect of H. pylori components on DC maturation. A total of 10 6 DCs/ml were stimulated with viable H. pylori strains or H. pylori sonicate (100 μg/ml) for 72 h. Controls included DCs treated with <t>PBS</t> (negative control) and E. coli <t>LPS</t> at a concentration
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bsa
    Requirement of CXCR4 but not CXCR7 for SDF-1-induced migration of <t>EPCs.</t> EPC migration was assayed in 24-transwell culture plates containing microporous (8.0 μM) membranes. (A) Dose–response assay, for which EPCs suspended in EBM-2 medium supplemented with 0.5% <t>BSA</t> were added to the top chamber, and SDF-1 was added to the low chamber at a concentrations of 0, 1, 10 and 100 ng/ml in EBM-2 medium supplemented with 1% FBS. (B) Effect of CXCR7 or CXCR4 inhibition on the SDF-1-induced migration of EPCs, for which EPCs suspended in the above mentioned medium were added to the top chamber in presence of anti-CXCR4 antibody (α CXCR4), anti-CXCR7 antibody (α CXCR7), IgG control, AMD3100 or CCX733, and SDF-1 at a concentration of 10 ng/ml in EBM-2 medium supplemented with 1% FBS was added to the low chamber. Results are given as mean ± S.D. of three independent experiments (* P
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e coli bl21
    Requirement of CXCR4 but not CXCR7 for SDF-1-induced migration of <t>EPCs.</t> EPC migration was assayed in 24-transwell culture plates containing microporous (8.0 μM) membranes. (A) Dose–response assay, for which EPCs suspended in EBM-2 medium supplemented with 0.5% <t>BSA</t> were added to the top chamber, and SDF-1 was added to the low chamber at a concentrations of 0, 1, 10 and 100 ng/ml in EBM-2 medium supplemented with 1% FBS. (B) Effect of CXCR7 or CXCR4 inhibition on the SDF-1-induced migration of EPCs, for which EPCs suspended in the above mentioned medium were added to the top chamber in presence of anti-CXCR4 antibody (α CXCR4), anti-CXCR7 antibody (α CXCR7), IgG control, AMD3100 or CCX733, and SDF-1 at a concentration of 10 ng/ml in EBM-2 medium supplemented with 1% FBS was added to the low chamber. Results are given as mean ± S.D. of three independent experiments (* P
    E Coli Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fibronectin
    TβRIII/β-arrestin2 regulates FN fibrillogenesis and incorporation of integrin α5β1 into sites of focal adhesions (A) Fluorescent microscopy analysis of endogenous FN fibrils in indicated cell lines using <t>anti-fibronectin</t> antibody. Bar= 3.5μm. (B) TIRF microscopy of MCF10A shTβRIII, control shNTC cells or shTβRIII cells transfected with rat TβRIII cells plated on FN coated coverslips, labeled with anti-vinculin and anti-α5 antibody, Bar=25μm. (C) Quantitation of colocalization of endogenous integrin α5 and vinculin was performed as described in Methods and is presented below (*p
    Fibronectin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore l glutamine
    TβRIII/β-arrestin2 regulates FN fibrillogenesis and incorporation of integrin α5β1 into sites of focal adhesions (A) Fluorescent microscopy analysis of endogenous FN fibrils in indicated cell lines using <t>anti-fibronectin</t> antibody. Bar= 3.5μm. (B) TIRF microscopy of MCF10A shTβRIII, control shNTC cells or shTβRIII cells transfected with rat TβRIII cells plated on FN coated coverslips, labeled with anti-vinculin and anti-α5 antibody, Bar=25μm. (C) Quantitation of colocalization of endogenous integrin α5 and vinculin was performed as described in Methods and is presented below (*p
    L Glutamine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 52942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore poly l lysine
    OPC maturation on MMP7‐cleaved plasma fibronectin, cellular fibronectin and fibronectin aggregates. Oligodendrocyte progenitor cells (OPCs) were differentiated on poly‐ l ‐lysine <t>(PLL),</t> intact plasma fibronectin (pFn), cellular fibronectin (cFn), fibronectin aggregates (aFn) or MMP7‐degraded plasma fibronectin (pFn‐MMP7), cellular Fn (cFn + MMP7) or fibronectin aggregates (aFn‐MMP7) for 6 days. The experiments on the structural variants are performed in independent experiments, while PLL is used as a positive control in each experiment. (a and b) Representative overview (a) and single cell (b) images of MBP immunocytochemistry (red) of oligodendrocytes (OLGs, 6 days in differentiation) cultured on the indicates substrates. DAPI‐stained nuclei are indicated in blue. Scale bar are 75 μm (a) and 10 μm (b). (c and d) The percentage of MBP‐positive cells (c) and the percentage of MBP‐positive cells bearing myelin membranes (d) were assessed 6 days after initiating differentiation. Bars depict mean + SEM of at least five independent experiments. In each experiment, the data of cells cultured on PLL was set at 100% (horizontal line). The percentages of MBP‐positive cells or myelin membranes in cells cultured on PLL were respectively 33.6.8% ± 9.5% and 24.0% ± 10.2% for the pFn‐related experiments, 34.9.8% ± 8.6% and 26.1% ± 8.2% for the cFn‐related experiments, and 43.8% ± 11.9% and 29.1% ± 8.7% for the aFn‐related experiments. Statistical differences with cells cultured on PLL (one sample t test, *** p
    Poly L Lysine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore chloramphenicol
    OPC maturation on MMP7‐cleaved plasma fibronectin, cellular fibronectin and fibronectin aggregates. Oligodendrocyte progenitor cells (OPCs) were differentiated on poly‐ l ‐lysine <t>(PLL),</t> intact plasma fibronectin (pFn), cellular fibronectin (cFn), fibronectin aggregates (aFn) or MMP7‐degraded plasma fibronectin (pFn‐MMP7), cellular Fn (cFn + MMP7) or fibronectin aggregates (aFn‐MMP7) for 6 days. The experiments on the structural variants are performed in independent experiments, while PLL is used as a positive control in each experiment. (a and b) Representative overview (a) and single cell (b) images of MBP immunocytochemistry (red) of oligodendrocytes (OLGs, 6 days in differentiation) cultured on the indicates substrates. DAPI‐stained nuclei are indicated in blue. Scale bar are 75 μm (a) and 10 μm (b). (c and d) The percentage of MBP‐positive cells (c) and the percentage of MBP‐positive cells bearing myelin membranes (d) were assessed 6 days after initiating differentiation. Bars depict mean + SEM of at least five independent experiments. In each experiment, the data of cells cultured on PLL was set at 100% (horizontal line). The percentages of MBP‐positive cells or myelin membranes in cells cultured on PLL were respectively 33.6.8% ± 9.5% and 24.0% ± 10.2% for the pFn‐related experiments, 34.9.8% ± 8.6% and 26.1% ± 8.2% for the cFn‐related experiments, and 43.8% ± 11.9% and 29.1% ± 8.7% for the aFn‐related experiments. Statistical differences with cells cultured on PLL (one sample t test, *** p
    Chloramphenicol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bl21
    OPC maturation on MMP7‐cleaved plasma fibronectin, cellular fibronectin and fibronectin aggregates. Oligodendrocyte progenitor cells (OPCs) were differentiated on poly‐ l ‐lysine <t>(PLL),</t> intact plasma fibronectin (pFn), cellular fibronectin (cFn), fibronectin aggregates (aFn) or MMP7‐degraded plasma fibronectin (pFn‐MMP7), cellular Fn (cFn + MMP7) or fibronectin aggregates (aFn‐MMP7) for 6 days. The experiments on the structural variants are performed in independent experiments, while PLL is used as a positive control in each experiment. (a and b) Representative overview (a) and single cell (b) images of MBP immunocytochemistry (red) of oligodendrocytes (OLGs, 6 days in differentiation) cultured on the indicates substrates. DAPI‐stained nuclei are indicated in blue. Scale bar are 75 μm (a) and 10 μm (b). (c and d) The percentage of MBP‐positive cells (c) and the percentage of MBP‐positive cells bearing myelin membranes (d) were assessed 6 days after initiating differentiation. Bars depict mean + SEM of at least five independent experiments. In each experiment, the data of cells cultured on PLL was set at 100% (horizontal line). The percentages of MBP‐positive cells or myelin membranes in cells cultured on PLL were respectively 33.6.8% ± 9.5% and 24.0% ± 10.2% for the pFn‐related experiments, 34.9.8% ± 8.6% and 26.1% ± 8.2% for the cFn‐related experiments, and 43.8% ± 11.9% and 29.1% ± 8.7% for the aFn‐related experiments. Statistical differences with cells cultured on PLL (one sample t test, *** p
    Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore iodixanol
    Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular and aortic endothelial cells. (a) Nanoparticle tracking analysis (NTA) of PC3 sEVs. (b) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions) in TCL and sEV lysates from PC3 cells; right panel, expression of ALIX and TSG101 (reducing conditions) in TCL and sEV lysates from PC3 cells. (c) Quantification of β6 mRNA expression by q-PCR in PC3 cells (positive control for β6 mRNA expression) and BAEC treated with αvβ6-positive PC3 sEVs for 4, 8 and 16 h or PBS vehicle control for 16 h. The GAPDH levels were comparable in PC3 and BAEC. The β6 mRNA expression is normalised to GAPDH. (d) Quantification of β6 mRNA expression by q-PCR in PC3 cells and HMEC1 treated with αvβ6-positive PC3 sEVs for 2, 4, 8, 16 and 24 h or PBS vehicle control for 24 h. The GAPDH levels were comparable in PC3 and HMEC1. The β6 mRNA expression is normalised to GAPDH. (e) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS or PC3 sEVs for the indicated time lengths (6 and 24 h) followed by IB analysis of TCL under non-reducing conditions for expression of β6 and CANX (loading control). (f) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS, or <t>iodixanol</t> density gradient separated PC3 sEVs for indicated time lengths (6, 16 and 24 h) followed by IB analysis for expression of β6 integrin subunit and CANX (loading control) in TCL under non-reducing conditions. (g) FACS analysis of cell-surface expression of αvβ6 integrin on HMEC1 incubated with PBS or PC3 sEVs for 18 h. The shift in fluorescence intensity for positive control PC3 cells (red line) or HMEC1 incubated with PC3 sEVs (blue line) show cell-surface expression of αvβ6 integrin compared to the isotype control (green line) and HMEC1 incubated with PBS-only (orange line). Different gels were used to separate samples under reducing or non- reducing conditions.
    Iodixanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1366 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dtt
    Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular and aortic endothelial cells. (a) Nanoparticle tracking analysis (NTA) of PC3 sEVs. (b) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions) in TCL and sEV lysates from PC3 cells; right panel, expression of ALIX and TSG101 (reducing conditions) in TCL and sEV lysates from PC3 cells. (c) Quantification of β6 mRNA expression by q-PCR in PC3 cells (positive control for β6 mRNA expression) and BAEC treated with αvβ6-positive PC3 sEVs for 4, 8 and 16 h or PBS vehicle control for 16 h. The GAPDH levels were comparable in PC3 and BAEC. The β6 mRNA expression is normalised to GAPDH. (d) Quantification of β6 mRNA expression by q-PCR in PC3 cells and HMEC1 treated with αvβ6-positive PC3 sEVs for 2, 4, 8, 16 and 24 h or PBS vehicle control for 24 h. The GAPDH levels were comparable in PC3 and HMEC1. The β6 mRNA expression is normalised to GAPDH. (e) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS or PC3 sEVs for the indicated time lengths (6 and 24 h) followed by IB analysis of TCL under non-reducing conditions for expression of β6 and CANX (loading control). (f) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS, or <t>iodixanol</t> density gradient separated PC3 sEVs for indicated time lengths (6, 16 and 24 h) followed by IB analysis for expression of β6 integrin subunit and CANX (loading control) in TCL under non-reducing conditions. (g) FACS analysis of cell-surface expression of αvβ6 integrin on HMEC1 incubated with PBS or PC3 sEVs for 18 h. The shift in fluorescence intensity for positive control PC3 cells (red line) or HMEC1 incubated with PC3 sEVs (blue line) show cell-surface expression of αvβ6 integrin compared to the isotype control (green line) and HMEC1 incubated with PBS-only (orange line). Different gels were used to separate samples under reducing or non- reducing conditions.
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    Image Search Results


    Localisation of galectin-3 and hnRNPA2B1 in cell nuclei. a HeLa cells were fixed and stained by immunofluorescence with anti-galectin-3/ Alexa Fluor 546 together with anti-hnRNPA2B1/ Alexa Fluor 647. Punctate structures positive for hnRNPA2B1 and endogenous galectin-3 are indicated by arrows. Nuclear staining (DAPI) is depicted in blue, scale bars: 10 μm. b Interaction between galectin-3 and hnRNPA2B1 was assessed by in situ PLA. HeLa cells were fixed and incubated with antibodies directed against galectin-3 and hnRNPA2B1. In the negative control HeLa cells were incubated in the absence of primary antibodies. HeLa cells were incubated with primary antibodies against hnRNPA2B1 and Sc35 as positive control. Interactions in a proximity up to 40 nm appear as fluorescent dots. Nuclei were stained with DAPI. For a better comparison, nuclei are depicted in blue, cytoplasmic PLA-signals in magenta and nuclear PLA-signals in dark blue in the merged images. Scale bars; 10 μm. c The amount of PLA-spots per nucleus was quantified. Bar graphs indicate the average relative number of PLA-signals per nucleus +/− SD, n = 3 (** p = 0.01)

    Journal: BMC Cancer

    Article Title: Galectin-3 interacts with components of the nuclear ribonucleoprotein complex

    doi: 10.1186/s12885-016-2546-0

    Figure Lengend Snippet: Localisation of galectin-3 and hnRNPA2B1 in cell nuclei. a HeLa cells were fixed and stained by immunofluorescence with anti-galectin-3/ Alexa Fluor 546 together with anti-hnRNPA2B1/ Alexa Fluor 647. Punctate structures positive for hnRNPA2B1 and endogenous galectin-3 are indicated by arrows. Nuclear staining (DAPI) is depicted in blue, scale bars: 10 μm. b Interaction between galectin-3 and hnRNPA2B1 was assessed by in situ PLA. HeLa cells were fixed and incubated with antibodies directed against galectin-3 and hnRNPA2B1. In the negative control HeLa cells were incubated in the absence of primary antibodies. HeLa cells were incubated with primary antibodies against hnRNPA2B1 and Sc35 as positive control. Interactions in a proximity up to 40 nm appear as fluorescent dots. Nuclei were stained with DAPI. For a better comparison, nuclei are depicted in blue, cytoplasmic PLA-signals in magenta and nuclear PLA-signals in dark blue in the merged images. Scale bars; 10 μm. c The amount of PLA-spots per nucleus was quantified. Bar graphs indicate the average relative number of PLA-signals per nucleus +/− SD, n = 3 (** p = 0.01)

    Article Snippet: Co-immunoprecipitation and BN-PAGE Nuclear extracts (NE) from RCC FG1 cells and HeLa cells were prepared with NE-PER nuclear and cytoplasmic extraction reagents-kit obtained from Thermo Scientific, Dreieich, Germany.

    Techniques: Staining, Immunofluorescence, In Situ, Proximity Ligation Assay, Incubation, Negative Control, Positive Control

    Activities of creatinase expressed in spore wall deficient mutants, osw2Δ, dit1Δ , and chs3Δ . (A) 50 µg of lysates from wild-type (wt), osw2Δ, dit1Δ , or chs3Δ spores expressing creatinase–HA were subjected to western blot analysis using an anti–HA antibody to detect the HA fusion (arrow). Wild-type spores harboring the empty vector was used as a control. (B, C) Creatinase activity was measured for 28 mg of indicated wet spores (B) or 5 mg of freeze-dried spores (C) expressing creatinase–HA. Spore samples were purified by percoll gradient centrifugation before measuring weight or freeze-drying. Wild-type spores harboring the empty vector were used as a control. Data presented are the mean ± SE of 3 independent samples. * P

    Journal: Bioengineered

    Article Title: Production of encapsulated creatinase using yeast spores

    doi: 10.1080/21655979.2016.1241926

    Figure Lengend Snippet: Activities of creatinase expressed in spore wall deficient mutants, osw2Δ, dit1Δ , and chs3Δ . (A) 50 µg of lysates from wild-type (wt), osw2Δ, dit1Δ , or chs3Δ spores expressing creatinase–HA were subjected to western blot analysis using an anti–HA antibody to detect the HA fusion (arrow). Wild-type spores harboring the empty vector was used as a control. (B, C) Creatinase activity was measured for 28 mg of indicated wet spores (B) or 5 mg of freeze-dried spores (C) expressing creatinase–HA. Spore samples were purified by percoll gradient centrifugation before measuring weight or freeze-drying. Wild-type spores harboring the empty vector were used as a control. Data presented are the mean ± SE of 3 independent samples. * P

    Article Snippet: After the washes, the resulting pellet was resuspended in 1 mL of 0.5% v/v Triton-X and layered on top of Percoll (Sigma-Aldrich, Shanghai, China) gradients (50-80% v/v Percoll, 10% v/v 2.5 M sucrose and 0.5% v/v Triton-X).

    Techniques: Expressing, Western Blot, Plasmid Preparation, Activity Assay, Purification, Gradient Centrifugation

    The bulk of milk microRNA pellets at low ultracentrifugation speeds and microRNAs distribution do not correspond to EV-associated proteins profiles. (a) Commercial milk preparation (100 mL) was subjected to successive differential ultracentrifugation steps at 12,000 g , 35,000 g , 70,000 g and 100,000 g for 1 h each at 4°C, and each pellet was kept and suspended in 1 mL PBS containing EDTA, as detailed in the Material and methods section. (b) Each sample (250 µL) was mixed with 750 µL of TRIzol-LS reagent for total RNA isolation and subsequent RT-qPCR detection of microRNAs bta-miR-223, bta-miR-125b, bta-miR-148a, bta-miR-29b, bta-miR-151-3p and bta-miR-2478. The experiment was performed three times with three different milk samples, and each quantification level in each pellet was reported on the sum of all the pellet (total) and expressed as a percentage of the total (mean ± SD; n = 3 or 6). The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post hoc comparison of the means with Tukey’s correction with p

    Journal: Journal of Extracellular Vesicles

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

    doi: 10.1080/20013078.2017.1401897

    Figure Lengend Snippet: The bulk of milk microRNA pellets at low ultracentrifugation speeds and microRNAs distribution do not correspond to EV-associated proteins profiles. (a) Commercial milk preparation (100 mL) was subjected to successive differential ultracentrifugation steps at 12,000 g , 35,000 g , 70,000 g and 100,000 g for 1 h each at 4°C, and each pellet was kept and suspended in 1 mL PBS containing EDTA, as detailed in the Material and methods section. (b) Each sample (250 µL) was mixed with 750 µL of TRIzol-LS reagent for total RNA isolation and subsequent RT-qPCR detection of microRNAs bta-miR-223, bta-miR-125b, bta-miR-148a, bta-miR-29b, bta-miR-151-3p and bta-miR-2478. The experiment was performed three times with three different milk samples, and each quantification level in each pellet was reported on the sum of all the pellet (total) and expressed as a percentage of the total (mean ± SD; n = 3 or 6). The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post hoc comparison of the means with Tukey’s correction with p

    Article Snippet: After each step, the pellets were resuspended in 1 mL of 0.22 µm filtered sterile phosphate-buffered saline (PBS) containing 100 nM ethylenediaminetetraacetic acid (EDTA) pH 7.4, and either (1) stored at −80°C for subsequent pellet analysis or (2) immediately diluted in 3 volumes of sterile PBS containing 100 nM EDTA, prior to layering on top of a 10–40% Iodixanol (Optiprep; Sigma-Aldrich, Oakville, ON, CA; in PBS) density gradient (IDG).

    Techniques: Isolation, Quantitative RT-PCR

    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env −  and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and  gag  with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env −  and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the  gag  precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).

    Journal: Journal of Virology

    Article Title: Human Immunodeficiency Virus Type 1 Envelope Glycoproteins That Lack Cytoplasmic Domain Cysteines: Impact on Association with Membrane Lipid Rafts and Incorporation onto Budding Virus Particles

    doi: 10.1128/JVI.78.10.5500-5506.2004

    Figure Lengend Snippet: Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env − and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and gag with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env − and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the gag precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).

    Article Snippet: Transfected cells were lysed with Triton X-100 at a final concentration of 0.5% in TNE (10 mM Tris, 100 mM sodium chloride, 10 mM EGTA [pH 7.5], protease inhibitor cocktail [Sigma Inc.]) for 30 min. Lysates were homogenized and centrifuged at low speed to remove nuclei.

    Techniques: Mutagenesis, Western Blot, Fractionation, Marker, Transduction

    Effect of H. pylori components on DC maturation. A total of 10 6 DCs/ml were stimulated with viable H. pylori strains or H. pylori sonicate (100 μg/ml) for 72 h. Controls included DCs treated with PBS (negative control) and E. coli LPS at a concentration

    Journal:

    Article Title: Impact of Helicobacter pylori Virulence Factors and Compounds on Activation and Maturation of Human Dendritic Cells

    doi: 10.1128/IAI.73.7.4180-4189.2005

    Figure Lengend Snippet: Effect of H. pylori components on DC maturation. A total of 10 6 DCs/ml were stimulated with viable H. pylori strains or H. pylori sonicate (100 μg/ml) for 72 h. Controls included DCs treated with PBS (negative control) and E. coli LPS at a concentration

    Article Snippet: Cells were either stimulated with 10 μl PBS, 100 ng/ml E. coli LPS 055:B5 (Sigma-Aldrich, Taufkirchen, Germany), or different H. pylori strains (formalin- or heat-inactivated H. pylori strains, as well as sonicate).

    Techniques: Negative Control, Concentration Assay

    Effects of different H. pylori strains on DC maturation. A total of 10 6 DCs/ml were stimulated with different H. pylori strains (MOI, 10) for 72 h. Controls included DCs treated with PBS (negative control) and E. coli- LPS at a concentration of 100 ng/ml

    Journal:

    Article Title: Impact of Helicobacter pylori Virulence Factors and Compounds on Activation and Maturation of Human Dendritic Cells

    doi: 10.1128/IAI.73.7.4180-4189.2005

    Figure Lengend Snippet: Effects of different H. pylori strains on DC maturation. A total of 10 6 DCs/ml were stimulated with different H. pylori strains (MOI, 10) for 72 h. Controls included DCs treated with PBS (negative control) and E. coli- LPS at a concentration of 100 ng/ml

    Article Snippet: Cells were either stimulated with 10 μl PBS, 100 ng/ml E. coli LPS 055:B5 (Sigma-Aldrich, Taufkirchen, Germany), or different H. pylori strains (formalin- or heat-inactivated H. pylori strains, as well as sonicate).

    Techniques: Negative Control, Concentration Assay

    Cytokines released by DCs stimulated with H. pylori . A total of 10 6 DCs/ml were pulsed with different H. pylori strains (MOI, 10) for 24 h. Controls included DCs stimulated with PBS (negative control) and E. coli- LPS at a concentration of 100 ng/ml (positive

    Journal:

    Article Title: Impact of Helicobacter pylori Virulence Factors and Compounds on Activation and Maturation of Human Dendritic Cells

    doi: 10.1128/IAI.73.7.4180-4189.2005

    Figure Lengend Snippet: Cytokines released by DCs stimulated with H. pylori . A total of 10 6 DCs/ml were pulsed with different H. pylori strains (MOI, 10) for 24 h. Controls included DCs stimulated with PBS (negative control) and E. coli- LPS at a concentration of 100 ng/ml (positive

    Article Snippet: Cells were either stimulated with 10 μl PBS, 100 ng/ml E. coli LPS 055:B5 (Sigma-Aldrich, Taufkirchen, Germany), or different H. pylori strains (formalin- or heat-inactivated H. pylori strains, as well as sonicate).

    Techniques: Negative Control, Concentration Assay

    Stimulatory ability of DC in the MLR assay. DCs were treated with H. pylori , E. coli- LPS (positive control), and PBS (negative control) for 24 h. DCs were harvested and washed twice with PBS before being cocultured with allogeneic T cells (5 ×

    Journal:

    Article Title: Impact of Helicobacter pylori Virulence Factors and Compounds on Activation and Maturation of Human Dendritic Cells

    doi: 10.1128/IAI.73.7.4180-4189.2005

    Figure Lengend Snippet: Stimulatory ability of DC in the MLR assay. DCs were treated with H. pylori , E. coli- LPS (positive control), and PBS (negative control) for 24 h. DCs were harvested and washed twice with PBS before being cocultured with allogeneic T cells (5 ×

    Article Snippet: Cells were either stimulated with 10 μl PBS, 100 ng/ml E. coli LPS 055:B5 (Sigma-Aldrich, Taufkirchen, Germany), or different H. pylori strains (formalin- or heat-inactivated H. pylori strains, as well as sonicate).

    Techniques: Mlr Assay, Positive Control, Negative Control

    Requirement of CXCR4 but not CXCR7 for SDF-1-induced migration of EPCs. EPC migration was assayed in 24-transwell culture plates containing microporous (8.0 μM) membranes. (A) Dose–response assay, for which EPCs suspended in EBM-2 medium supplemented with 0.5% BSA were added to the top chamber, and SDF-1 was added to the low chamber at a concentrations of 0, 1, 10 and 100 ng/ml in EBM-2 medium supplemented with 1% FBS. (B) Effect of CXCR7 or CXCR4 inhibition on the SDF-1-induced migration of EPCs, for which EPCs suspended in the above mentioned medium were added to the top chamber in presence of anti-CXCR4 antibody (α CXCR4), anti-CXCR7 antibody (α CXCR7), IgG control, AMD3100 or CCX733, and SDF-1 at a concentration of 10 ng/ml in EBM-2 medium supplemented with 1% FBS was added to the low chamber. Results are given as mean ± S.D. of three independent experiments (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The role of CXCR7 on the adhesion, proliferation and angiogenesis of endothelial progenitor cells

    doi: 10.1111/j.1582-4934.2011.01301.x

    Figure Lengend Snippet: Requirement of CXCR4 but not CXCR7 for SDF-1-induced migration of EPCs. EPC migration was assayed in 24-transwell culture plates containing microporous (8.0 μM) membranes. (A) Dose–response assay, for which EPCs suspended in EBM-2 medium supplemented with 0.5% BSA were added to the top chamber, and SDF-1 was added to the low chamber at a concentrations of 0, 1, 10 and 100 ng/ml in EBM-2 medium supplemented with 1% FBS. (B) Effect of CXCR7 or CXCR4 inhibition on the SDF-1-induced migration of EPCs, for which EPCs suspended in the above mentioned medium were added to the top chamber in presence of anti-CXCR4 antibody (α CXCR4), anti-CXCR7 antibody (α CXCR7), IgG control, AMD3100 or CCX733, and SDF-1 at a concentration of 10 ng/ml in EBM-2 medium supplemented with 1% FBS was added to the low chamber. Results are given as mean ± S.D. of three independent experiments (* P

    Article Snippet: Briefly, EPCs suspended in EBM-2 medium supplemented with 0.5% BSA (Sigma) were added to the top chamber.

    Techniques: Migration, Inhibition, Concentration Assay

    TβRIII/β-arrestin2 regulates FN fibrillogenesis and incorporation of integrin α5β1 into sites of focal adhesions (A) Fluorescent microscopy analysis of endogenous FN fibrils in indicated cell lines using anti-fibronectin antibody. Bar= 3.5μm. (B) TIRF microscopy of MCF10A shTβRIII, control shNTC cells or shTβRIII cells transfected with rat TβRIII cells plated on FN coated coverslips, labeled with anti-vinculin and anti-α5 antibody, Bar=25μm. (C) Quantitation of colocalization of endogenous integrin α5 and vinculin was performed as described in Methods and is presented below (*p

    Journal: Oncogene

    Article Title: T?RIII/?-arrestin2 regulates integrin ?5?1 trafficking, function, and localization in epithelial cells

    doi: 10.1038/onc.2012.157

    Figure Lengend Snippet: TβRIII/β-arrestin2 regulates FN fibrillogenesis and incorporation of integrin α5β1 into sites of focal adhesions (A) Fluorescent microscopy analysis of endogenous FN fibrils in indicated cell lines using anti-fibronectin antibody. Bar= 3.5μm. (B) TIRF microscopy of MCF10A shTβRIII, control shNTC cells or shTβRIII cells transfected with rat TβRIII cells plated on FN coated coverslips, labeled with anti-vinculin and anti-α5 antibody, Bar=25μm. (C) Quantitation of colocalization of endogenous integrin α5 and vinculin was performed as described in Methods and is presented below (*p

    Article Snippet: Thirty-five mm glass bottom culture dishes (MatTek) coated with 5μg/mL fibronectin (Calbiochem) were used to image fixed cells on a Leica AM TIRF microscope.

    Techniques: Microscopy, Transfection, Labeling, Quantitation Assay

    TβRIII mediates epithelial cell adhesion to fibronectin, regulates focal adhesion formation and FAK activation in response to spreading on FN MCF10A mammary epithelial cells adenovirally infected with either shTβRIII-1 or shTβRIII-2 to human TβRIII in parallel with controls (shNTC or shCtl) were (A) assessed for cell surface expression of TβRIII by 125 I-TGF-β binding and crosslinking, and for total cellular β-arrestin2, fibronectin and integrin α5 expression, with β-actin as a loading control, and (B) assessed for adhesion to the indicated concentrations of FN for 30 minutes and the adhesion level plotted (mean+/−SD ,*p

    Journal: Oncogene

    Article Title: T?RIII/?-arrestin2 regulates integrin ?5?1 trafficking, function, and localization in epithelial cells

    doi: 10.1038/onc.2012.157

    Figure Lengend Snippet: TβRIII mediates epithelial cell adhesion to fibronectin, regulates focal adhesion formation and FAK activation in response to spreading on FN MCF10A mammary epithelial cells adenovirally infected with either shTβRIII-1 or shTβRIII-2 to human TβRIII in parallel with controls (shNTC or shCtl) were (A) assessed for cell surface expression of TβRIII by 125 I-TGF-β binding and crosslinking, and for total cellular β-arrestin2, fibronectin and integrin α5 expression, with β-actin as a loading control, and (B) assessed for adhesion to the indicated concentrations of FN for 30 minutes and the adhesion level plotted (mean+/−SD ,*p

    Article Snippet: Thirty-five mm glass bottom culture dishes (MatTek) coated with 5μg/mL fibronectin (Calbiochem) were used to image fixed cells on a Leica AM TIRF microscope.

    Techniques: Activation Assay, Infection, Expressing, Binding Assay

    OPC maturation on MMP7‐cleaved plasma fibronectin, cellular fibronectin and fibronectin aggregates. Oligodendrocyte progenitor cells (OPCs) were differentiated on poly‐ l ‐lysine (PLL), intact plasma fibronectin (pFn), cellular fibronectin (cFn), fibronectin aggregates (aFn) or MMP7‐degraded plasma fibronectin (pFn‐MMP7), cellular Fn (cFn + MMP7) or fibronectin aggregates (aFn‐MMP7) for 6 days. The experiments on the structural variants are performed in independent experiments, while PLL is used as a positive control in each experiment. (a and b) Representative overview (a) and single cell (b) images of MBP immunocytochemistry (red) of oligodendrocytes (OLGs, 6 days in differentiation) cultured on the indicates substrates. DAPI‐stained nuclei are indicated in blue. Scale bar are 75 μm (a) and 10 μm (b). (c and d) The percentage of MBP‐positive cells (c) and the percentage of MBP‐positive cells bearing myelin membranes (d) were assessed 6 days after initiating differentiation. Bars depict mean + SEM of at least five independent experiments. In each experiment, the data of cells cultured on PLL was set at 100% (horizontal line). The percentages of MBP‐positive cells or myelin membranes in cells cultured on PLL were respectively 33.6.8% ± 9.5% and 24.0% ± 10.2% for the pFn‐related experiments, 34.9.8% ± 8.6% and 26.1% ± 8.2% for the cFn‐related experiments, and 43.8% ± 11.9% and 29.1% ± 8.7% for the aFn‐related experiments. Statistical differences with cells cultured on PLL (one sample t test, *** p

    Journal: Glia

    Article Title: MMP7 cleaves remyelination‐impairing fibronectin aggregates and its expression is reduced in chronic multiple sclerosis lesions, et al. MMP7 cleaves remyelination‐impairing fibronectin aggregates and its expression is reduced in chronic multiple sclerosis lesions

    doi: 10.1002/glia.23328

    Figure Lengend Snippet: OPC maturation on MMP7‐cleaved plasma fibronectin, cellular fibronectin and fibronectin aggregates. Oligodendrocyte progenitor cells (OPCs) were differentiated on poly‐ l ‐lysine (PLL), intact plasma fibronectin (pFn), cellular fibronectin (cFn), fibronectin aggregates (aFn) or MMP7‐degraded plasma fibronectin (pFn‐MMP7), cellular Fn (cFn + MMP7) or fibronectin aggregates (aFn‐MMP7) for 6 days. The experiments on the structural variants are performed in independent experiments, while PLL is used as a positive control in each experiment. (a and b) Representative overview (a) and single cell (b) images of MBP immunocytochemistry (red) of oligodendrocytes (OLGs, 6 days in differentiation) cultured on the indicates substrates. DAPI‐stained nuclei are indicated in blue. Scale bar are 75 μm (a) and 10 μm (b). (c and d) The percentage of MBP‐positive cells (c) and the percentage of MBP‐positive cells bearing myelin membranes (d) were assessed 6 days after initiating differentiation. Bars depict mean + SEM of at least five independent experiments. In each experiment, the data of cells cultured on PLL was set at 100% (horizontal line). The percentages of MBP‐positive cells or myelin membranes in cells cultured on PLL were respectively 33.6.8% ± 9.5% and 24.0% ± 10.2% for the pFn‐related experiments, 34.9.8% ± 8.6% and 26.1% ± 8.2% for the cFn‐related experiments, and 43.8% ± 11.9% and 29.1% ± 8.7% for the aFn‐related experiments. Statistical differences with cells cultured on PLL (one sample t test, *** p

    Article Snippet: Cells were cultured for 10–12 days on poly‐l ‐lysine (PLL, 5 µg/ml, Sigma)‐coated tissue culture flasks (Nalge Nunc, Naperville, IL).

    Techniques: Positive Control, Immunocytochemistry, Cell Culture, Staining

    Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular and aortic endothelial cells. (a) Nanoparticle tracking analysis (NTA) of PC3 sEVs. (b) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions) in TCL and sEV lysates from PC3 cells; right panel, expression of ALIX and TSG101 (reducing conditions) in TCL and sEV lysates from PC3 cells. (c) Quantification of β6 mRNA expression by q-PCR in PC3 cells (positive control for β6 mRNA expression) and BAEC treated with αvβ6-positive PC3 sEVs for 4, 8 and 16 h or PBS vehicle control for 16 h. The GAPDH levels were comparable in PC3 and BAEC. The β6 mRNA expression is normalised to GAPDH. (d) Quantification of β6 mRNA expression by q-PCR in PC3 cells and HMEC1 treated with αvβ6-positive PC3 sEVs for 2, 4, 8, 16 and 24 h or PBS vehicle control for 24 h. The GAPDH levels were comparable in PC3 and HMEC1. The β6 mRNA expression is normalised to GAPDH. (e) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS or PC3 sEVs for the indicated time lengths (6 and 24 h) followed by IB analysis of TCL under non-reducing conditions for expression of β6 and CANX (loading control). (f) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS, or iodixanol density gradient separated PC3 sEVs for indicated time lengths (6, 16 and 24 h) followed by IB analysis for expression of β6 integrin subunit and CANX (loading control) in TCL under non-reducing conditions. (g) FACS analysis of cell-surface expression of αvβ6 integrin on HMEC1 incubated with PBS or PC3 sEVs for 18 h. The shift in fluorescence intensity for positive control PC3 cells (red line) or HMEC1 incubated with PC3 sEVs (blue line) show cell-surface expression of αvβ6 integrin compared to the isotype control (green line) and HMEC1 incubated with PBS-only (orange line). Different gels were used to separate samples under reducing or non- reducing conditions.

    Journal: Journal of Extracellular Vesicles

    Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

    doi: 10.1080/20013078.2020.1763594

    Figure Lengend Snippet: Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular and aortic endothelial cells. (a) Nanoparticle tracking analysis (NTA) of PC3 sEVs. (b) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions) in TCL and sEV lysates from PC3 cells; right panel, expression of ALIX and TSG101 (reducing conditions) in TCL and sEV lysates from PC3 cells. (c) Quantification of β6 mRNA expression by q-PCR in PC3 cells (positive control for β6 mRNA expression) and BAEC treated with αvβ6-positive PC3 sEVs for 4, 8 and 16 h or PBS vehicle control for 16 h. The GAPDH levels were comparable in PC3 and BAEC. The β6 mRNA expression is normalised to GAPDH. (d) Quantification of β6 mRNA expression by q-PCR in PC3 cells and HMEC1 treated with αvβ6-positive PC3 sEVs for 2, 4, 8, 16 and 24 h or PBS vehicle control for 24 h. The GAPDH levels were comparable in PC3 and HMEC1. The β6 mRNA expression is normalised to GAPDH. (e) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS or PC3 sEVs for the indicated time lengths (6 and 24 h) followed by IB analysis of TCL under non-reducing conditions for expression of β6 and CANX (loading control). (f) HMEC1 plated (2 × 10 5 ) in six-well plates were incubated with PBS, or iodixanol density gradient separated PC3 sEVs for indicated time lengths (6, 16 and 24 h) followed by IB analysis for expression of β6 integrin subunit and CANX (loading control) in TCL under non-reducing conditions. (g) FACS analysis of cell-surface expression of αvβ6 integrin on HMEC1 incubated with PBS or PC3 sEVs for 18 h. The shift in fluorescence intensity for positive control PC3 cells (red line) or HMEC1 incubated with PC3 sEVs (blue line) show cell-surface expression of αvβ6 integrin compared to the isotype control (green line) and HMEC1 incubated with PBS-only (orange line). Different gels were used to separate samples under reducing or non- reducing conditions.

    Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.

    Techniques: Derivative Assay, Expressing, Polymerase Chain Reaction, Positive Control, Incubation, FACS, Fluorescence

    Down-regulation of αvβ6 integrin in prostate cancer sEVs modulates the angiogenic potential of microvascular endothelial cells. HMEC1 were seeded (1.5 × 10 4 , replicates n = 3) on 96-well plates coated with Matrigel and incubated with PBS or iodixanol density gradient separated sEVs (0.3 × 10 9 vesicles) from PC3-shCtrl, -shβ6 or -shβ5 cells. The upper panel shows representative micrographs ( n = 6 different fields for each group) of tubes formed during tube formation assays on HMEC1 after 5 h incubation with respective sEV type. The lower panel shows dot plots representing the number of nodes, junctions and tubules formed by HMEC1 in each sEV incubation group relative to PBS ( n = 6 different fields for each group). Values are reported as mean ± SEM, * P

    Journal: Journal of Extracellular Vesicles

    Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

    doi: 10.1080/20013078.2020.1763594

    Figure Lengend Snippet: Down-regulation of αvβ6 integrin in prostate cancer sEVs modulates the angiogenic potential of microvascular endothelial cells. HMEC1 were seeded (1.5 × 10 4 , replicates n = 3) on 96-well plates coated with Matrigel and incubated with PBS or iodixanol density gradient separated sEVs (0.3 × 10 9 vesicles) from PC3-shCtrl, -shβ6 or -shβ5 cells. The upper panel shows representative micrographs ( n = 6 different fields for each group) of tubes formed during tube formation assays on HMEC1 after 5 h incubation with respective sEV type. The lower panel shows dot plots representing the number of nodes, junctions and tubules formed by HMEC1 in each sEV incubation group relative to PBS ( n = 6 different fields for each group). Values are reported as mean ± SEM, * P

    Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.

    Techniques: Incubation

    Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular endothelial cells regulates survivin levels. (a) HMEC1 were plated (2 × 10 5 ) in six-well plates and incubated with PBS or sEVs derived from PC3-shCtrl, -shβ6 or -shβ5 cells for 18 h and after incubation, the TCL were analysed by IB under reducing conditions for expression of pSTAT1(Y701), STAT1 and ACTIN (loading control). (b) HMEC1 were plated (2 × 10 5 ) in six-well plates and incubated with PBS or sEVs derived from PC3-shCtrl, -shβ6 or -shβ5 cells for 18 h and after incubation, the TCL were analysed by IB under reducing conditions for expression of survivin and ACTIN (loading control). (c) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions); right panel, IB analysis for expression of β5 integrin subunit, ALIX, TSG101, CD9, survivin and CANX (reducing conditions) in TCL and sEV lysates from PC3 cells. (d) Iodixanol density gradient analysis of PC3 sEVs was performed as described in the Materials and methods. Expression of ALIX, TSG101, survivin, CANX (reducing conditions), CD63 and CD81 (non-reducing conditions) analysed by IB of 10 consecutive iodixanol density gradient fractions is shown. (e) IB analysis for expression of ALIX and survivin (reducing conditions) in TCL and sEV lysates from PC3-shCtrl and -shβ6 cells. Different gels were used to separate samples under reducing or non- reducing conditions.

    Journal: Journal of Extracellular Vesicles

    Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

    doi: 10.1080/20013078.2020.1763594

    Figure Lengend Snippet: Transfer of prostate cancer cell-derived αvβ6-positive sEVs to microvascular endothelial cells regulates survivin levels. (a) HMEC1 were plated (2 × 10 5 ) in six-well plates and incubated with PBS or sEVs derived from PC3-shCtrl, -shβ6 or -shβ5 cells for 18 h and after incubation, the TCL were analysed by IB under reducing conditions for expression of pSTAT1(Y701), STAT1 and ACTIN (loading control). (b) HMEC1 were plated (2 × 10 5 ) in six-well plates and incubated with PBS or sEVs derived from PC3-shCtrl, -shβ6 or -shβ5 cells for 18 h and after incubation, the TCL were analysed by IB under reducing conditions for expression of survivin and ACTIN (loading control). (c) Left panel, IB analysis for expression of β6 integrin subunit, CD63, CD81 and CANX (non-reducing conditions); right panel, IB analysis for expression of β5 integrin subunit, ALIX, TSG101, CD9, survivin and CANX (reducing conditions) in TCL and sEV lysates from PC3 cells. (d) Iodixanol density gradient analysis of PC3 sEVs was performed as described in the Materials and methods. Expression of ALIX, TSG101, survivin, CANX (reducing conditions), CD63 and CD81 (non-reducing conditions) analysed by IB of 10 consecutive iodixanol density gradient fractions is shown. (e) IB analysis for expression of ALIX and survivin (reducing conditions) in TCL and sEV lysates from PC3-shCtrl and -shβ6 cells. Different gels were used to separate samples under reducing or non- reducing conditions.

    Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.

    Techniques: Derivative Assay, Incubation, Expressing

    Characterisation of prostate cancer cell-derived αvβ6-positive LEVs and αvβ6-positive sEVs. (a) Iodixanol density gradient analysis of PC3 cell-derived large extracellular vesicles (PC3 LEVs) was performed as described in the Materials and methods. Expression of β6 integrin subunit, CD63 and CD81 analysed by immunoblotting (IB) (non-reducing conditions) in LEV lysates of 10 consecutive iodixanol density gradient fractions is shown. (b) Iodixanol density gradient analysis of PC3 cell-derived small extracellular vesicles (PC3 sEVs) was performed as described in the Materials and methods. IB analysis for expression of β6 integrin subunit, CD63 and CD81 (non-reducing conditions) and CD9 (reducing conditions) in sEV lysates of 10 consecutive iodixanol density gradient fractions is shown. IB of CANX (reducing conditions) in PC3-total cell lysates (PC3-TCL) and sEV lysates of 10 consecutive fractions is shown. Different gels were used to separate samples under reducing or non- reducing conditions

    Journal: Journal of Extracellular Vesicles

    Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

    doi: 10.1080/20013078.2020.1763594

    Figure Lengend Snippet: Characterisation of prostate cancer cell-derived αvβ6-positive LEVs and αvβ6-positive sEVs. (a) Iodixanol density gradient analysis of PC3 cell-derived large extracellular vesicles (PC3 LEVs) was performed as described in the Materials and methods. Expression of β6 integrin subunit, CD63 and CD81 analysed by immunoblotting (IB) (non-reducing conditions) in LEV lysates of 10 consecutive iodixanol density gradient fractions is shown. (b) Iodixanol density gradient analysis of PC3 cell-derived small extracellular vesicles (PC3 sEVs) was performed as described in the Materials and methods. IB analysis for expression of β6 integrin subunit, CD63 and CD81 (non-reducing conditions) and CD9 (reducing conditions) in sEV lysates of 10 consecutive iodixanol density gradient fractions is shown. IB of CANX (reducing conditions) in PC3-total cell lysates (PC3-TCL) and sEV lysates of 10 consecutive fractions is shown. Different gels were used to separate samples under reducing or non- reducing conditions

    Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.

    Techniques: Derivative Assay, Expressing

    Characterisation of prostate cancer cell-derived sEVs upon knockdown or expression of β6 integrin subunit in prostate cancer cells. (a) NTA of the sEV fraction (density 1.12 g/mL) from the iodixanol density gradient of PC3-shCtrl, PC3-shβ6 and PC3-shβ5 cell-derived sEVs. (b) NTA analysis of C4-2B-Mock or C4-2B-αvβ6-derived sEVs. (c) IB analysis for expression of β6 integrin subunit, CD63, CD81, (non-reducing conditions) and TSG101 (reducing conditions) in TCL and sEV lysates from PC3-shβ5, -shCtrl and -shβ6 cells. (d) IB analysis for expression of β6 integrin subunit, CANX (non-reducing conditions) and ALIX, TSG101, CD9 and CANX (reducing conditions) in TCL and sEV lysates from C4-2B-Mock or -αvβ6 cells. Different gels were used to separate samples under reducing or non- reducing conditions.

    Journal: Journal of Extracellular Vesicles

    Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

    doi: 10.1080/20013078.2020.1763594

    Figure Lengend Snippet: Characterisation of prostate cancer cell-derived sEVs upon knockdown or expression of β6 integrin subunit in prostate cancer cells. (a) NTA of the sEV fraction (density 1.12 g/mL) from the iodixanol density gradient of PC3-shCtrl, PC3-shβ6 and PC3-shβ5 cell-derived sEVs. (b) NTA analysis of C4-2B-Mock or C4-2B-αvβ6-derived sEVs. (c) IB analysis for expression of β6 integrin subunit, CD63, CD81, (non-reducing conditions) and TSG101 (reducing conditions) in TCL and sEV lysates from PC3-shβ5, -shCtrl and -shβ6 cells. (d) IB analysis for expression of β6 integrin subunit, CANX (non-reducing conditions) and ALIX, TSG101, CD9 and CANX (reducing conditions) in TCL and sEV lysates from C4-2B-Mock or -αvβ6 cells. Different gels were used to separate samples under reducing or non- reducing conditions.

    Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.

    Techniques: Derivative Assay, Expressing

    Expression of αvβ6 integrin in prostate cancer sEVs modulates the tube forming potential of endothelial cells. (a) HMEC1 were seeded (1.5 × 10 4 , replicates n = 3) on 96-well plates coated with Matrigel and incubated with PBS or iodixanol density gradient separated sEVs (0.3 × 10 9 vesicles) from C4-2B-Mock or -αvβ6 cells. The upper panel shows representative micrographs ( n = 6 different fields for each group) of tubes formed by HMEC1 after 5 h incubation with respective sEV type. The lower panel shows dot plots representing the number of nodes, junctions and tubules formed by HMEC1 in each sEV incubation group relative to PBS ( n = 6 different fields for each group). (b) BAEC were seeded (1.5 × 10 4 , replicates n = 3) on 96-well plates coated with Matrigel and incubated with PBS or iodixanol density gradient separated sEVs (0.3 × 10 9 vesicles) from C4-2B-Mock or -αvβ6 cells. Dot plots represent the number of nodes, junctions and tubules formed by BAEC 8 h after incubation with each sEV group relative to PBS ( n = 6 different fields for each group). Values are reported as mean ± SEM, * P

    Journal: Journal of Extracellular Vesicles

    Article Title: The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

    doi: 10.1080/20013078.2020.1763594

    Figure Lengend Snippet: Expression of αvβ6 integrin in prostate cancer sEVs modulates the tube forming potential of endothelial cells. (a) HMEC1 were seeded (1.5 × 10 4 , replicates n = 3) on 96-well plates coated with Matrigel and incubated with PBS or iodixanol density gradient separated sEVs (0.3 × 10 9 vesicles) from C4-2B-Mock or -αvβ6 cells. The upper panel shows representative micrographs ( n = 6 different fields for each group) of tubes formed by HMEC1 after 5 h incubation with respective sEV type. The lower panel shows dot plots representing the number of nodes, junctions and tubules formed by HMEC1 in each sEV incubation group relative to PBS ( n = 6 different fields for each group). (b) BAEC were seeded (1.5 × 10 4 , replicates n = 3) on 96-well plates coated with Matrigel and incubated with PBS or iodixanol density gradient separated sEVs (0.3 × 10 9 vesicles) from C4-2B-Mock or -αvβ6 cells. Dot plots represent the number of nodes, junctions and tubules formed by BAEC 8 h after incubation with each sEV group relative to PBS ( n = 6 different fields for each group). Values are reported as mean ± SEM, * P

    Article Snippet: Briefly, the LEVs obtained from PC3-parental cells or sEVs obtained from PC3 cells (PC3-parental, -shCtrl, -shβ5 and -shβ6) or C4-2B cells (C4-2B-Mock, -αvβ6) were suspended in 1.636 mL of 30% iodixanol solution [made by mixing 1:1 of 60% (wt/vol) stock solution of iodixanol (OptiPrep™, Sigma # 1556) with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, pH 7.4)] and layered at the bottom of an ultracentrifugation tube.

    Techniques: Expressing, Incubation