antibody expression  (Thermo Fisher)


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    Structured Review

    Thermo Fisher antibody expression
    Antibody Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody expression/product/Thermo Fisher
    Average 89 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    antibody expression - by Bioz Stars, 2020-09
    89/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer
    Article Snippet: .. Antibody expression and purification The tool antibody variable domains of anti-hCLDN18.2 antibody were synthesized at GeneArt or ATUM and cloned into mammalian expression vectors for full length hIgG2dA D265A that carry 223E, 225E, 228E, and 368E mutations. .. The anti-CD3 arm was cloned into hIgG2dA D265A vectors that carry 223R, 225R, 228R, and 409R mutations.

    Article Title: Characterization of a Macaque Recombinant Monoclonal Antibody That Binds to a CD4-Induced Epitope and Neutralizes Simian Immunodeficiency Virus
    Article Snippet: .. Individual CHO clones were isolated and screened for antibody expression by enzyme-linked immunosorbent assay (ELISA) for IgG in the culture supernatant using goat anti-human IgG to coat plates and alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Pierce, Rockford, Ill.) for detection. ..

    Amplification:

    Article Title: Systemic translocation of Staphylococcus drives autoantibody production in HIV disease
    Article Snippet: .. Single B cell immunoglobulin gene amplification and antibody expression As described in previous studies [ ], the frozen plates with sorted dsDNA-specific single B cells were thawed at room temperature, and reverse transcription was carried out by adding 3 μL of 50 μM random hexamers (Invitrogen), 1 μL of 10 mM dNTP mix (Invitrogen), and 1 μL of SuperScript III (Invitrogen) into each well. .. The mixture was incubated at 25 °C for 10 min, 50 °C for 50 min, and 85 °C for 5 min. IgH, Igκ, and Igλ genes were amplified from cDNA.

    Synthesized:

    Article Title: Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer
    Article Snippet: .. Antibody expression and purification The tool antibody variable domains of anti-hCLDN18.2 antibody were synthesized at GeneArt or ATUM and cloned into mammalian expression vectors for full length hIgG2dA D265A that carry 223E, 225E, 228E, and 368E mutations. .. The anti-CD3 arm was cloned into hIgG2dA D265A vectors that carry 223R, 225R, 228R, and 409R mutations.

    Isolation:

    Article Title: Characterization of a Macaque Recombinant Monoclonal Antibody That Binds to a CD4-Induced Epitope and Neutralizes Simian Immunodeficiency Virus
    Article Snippet: .. Individual CHO clones were isolated and screened for antibody expression by enzyme-linked immunosorbent assay (ELISA) for IgG in the culture supernatant using goat anti-human IgG to coat plates and alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Pierce, Rockford, Ill.) for detection. ..

    Purification:

    Article Title: Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer
    Article Snippet: .. Antibody expression and purification The tool antibody variable domains of anti-hCLDN18.2 antibody were synthesized at GeneArt or ATUM and cloned into mammalian expression vectors for full length hIgG2dA D265A that carry 223E, 225E, 228E, and 368E mutations. .. The anti-CD3 arm was cloned into hIgG2dA D265A vectors that carry 223R, 225R, 228R, and 409R mutations.

    Article Title: Novel conformation-specific monoclonal antibodies against amyloidogenic forms of transthyretin
    Article Snippet: .. Antibody expression and purification Hybridoma cells were expanded in shake flasks and seeded into 10–25 L Wave bag cultures (CD hybridoma expression media with Glutamax [Life Technology]). .. Batch cultures were prepared using a Wave Bioreactor (GE Healthcare, Piscataway, NJ; 37 °C, 7% CO2 under constant agitation) with periodic cell number monitoring.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Characterization of a Macaque Recombinant Monoclonal Antibody That Binds to a CD4-Induced Epitope and Neutralizes Simian Immunodeficiency Virus
    Article Snippet: .. Individual CHO clones were isolated and screened for antibody expression by enzyme-linked immunosorbent assay (ELISA) for IgG in the culture supernatant using goat anti-human IgG to coat plates and alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Pierce, Rockford, Ill.) for detection. ..

    Expressing:

    Article Title: Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer
    Article Snippet: .. Antibody expression and purification The tool antibody variable domains of anti-hCLDN18.2 antibody were synthesized at GeneArt or ATUM and cloned into mammalian expression vectors for full length hIgG2dA D265A that carry 223E, 225E, 228E, and 368E mutations. .. The anti-CD3 arm was cloned into hIgG2dA D265A vectors that carry 223R, 225R, 228R, and 409R mutations.

    Article Title: The Potent and Broadly Neutralizing Human Dengue Virus-Specific Monoclonal Antibody 1C19 Reveals a Unique Cross-Reactive Epitope on the bc Loop of Domain II of the Envelope Protein
    Article Snippet: .. For antibody expression, the cells in 75-cm2 flasks were collected with a cell scraper; the hybridomas were washed in serum-free medium (Gibco Hybridoma-SFM from Invitrogen; catalog no. 12045084) and split equally among four 225-cm2 flasks (Corning; catalog no. 431082) containing 250 ml serum-free medium. .. Antibodies were purified from clarified medium by protein G chromatography (GE Life Sciences; protein G HP columns).

    Article Title: Increased breadth of HIV-1 neutralization achieved by diverse antibody clones each with limited neutralization breadth
    Article Snippet: .. For antibody expression, the hybridomas in a 75-cm2 flasks were collected with a cell scraper, washed in serum-free medium (Gibco) and split equally among four 225-cm2 flasks (Corning) containing 250 mL of serum-free medium. ..

    Article Title: Novel conformation-specific monoclonal antibodies against amyloidogenic forms of transthyretin
    Article Snippet: .. Antibody expression and purification Hybridoma cells were expanded in shake flasks and seeded into 10–25 L Wave bag cultures (CD hybridoma expression media with Glutamax [Life Technology]). .. Batch cultures were prepared using a Wave Bioreactor (GE Healthcare, Piscataway, NJ; 37 °C, 7% CO2 under constant agitation) with periodic cell number monitoring.

    Article Title: Systemic translocation of Staphylococcus drives autoantibody production in HIV disease
    Article Snippet: .. Single B cell immunoglobulin gene amplification and antibody expression As described in previous studies [ ], the frozen plates with sorted dsDNA-specific single B cells were thawed at room temperature, and reverse transcription was carried out by adding 3 μL of 50 μM random hexamers (Invitrogen), 1 μL of 10 mM dNTP mix (Invitrogen), and 1 μL of SuperScript III (Invitrogen) into each well. .. The mixture was incubated at 25 °C for 10 min, 50 °C for 50 min, and 85 °C for 5 min. IgH, Igκ, and Igλ genes were amplified from cDNA.

    Article Title: Characterization of a Macaque Recombinant Monoclonal Antibody That Binds to a CD4-Induced Epitope and Neutralizes Simian Immunodeficiency Virus
    Article Snippet: .. Individual CHO clones were isolated and screened for antibody expression by enzyme-linked immunosorbent assay (ELISA) for IgG in the culture supernatant using goat anti-human IgG to coat plates and alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Pierce, Rockford, Ill.) for detection. ..

    Article Title: Expression of antibodies using single open reading frame (sORF) vector design
    Article Snippet: .. Western blot analysis For the analysis of intracellular polyprotein and antibody expression, cells were lysed by heating in 1X NuPAGE LDS Sample Buffer (Life Technologies, cat.# NP0007) with NuPAGE Sample Reducing Agent (Life Technologies, cat.# NP0004). .. Samples were separated on SDS-PAGE gel, transferred to nitrocellulose and blotted with antibodies against human IgG (Thermo Scientific, cat.# 31413), human kappa LC (Thermo Scientific, cat.# 31414) or human lambda LC (Thermo Scientific, cat.# 31131).

    Article Title: Recognition of influenza H3N2 variant virus by human neutralizing antibodies
    Article Snippet: .. The cells then were washed and expanded equally to four 225-cm2 flasks for antibody expression in serum-free medium (GIBCO Hybridoma-SFM, Invitrogen, 12045084). .. The supernatant was harvested after 3 weeks, filtered with a 0.4-μm filter, and the monoclonal IgGs were purified by affinity chromatography using protein G columns (GE Life Sciences, Protein G HP Columns).

    Western Blot:

    Article Title: Expression of antibodies using single open reading frame (sORF) vector design
    Article Snippet: .. Western blot analysis For the analysis of intracellular polyprotein and antibody expression, cells were lysed by heating in 1X NuPAGE LDS Sample Buffer (Life Technologies, cat.# NP0007) with NuPAGE Sample Reducing Agent (Life Technologies, cat.# NP0004). .. Samples were separated on SDS-PAGE gel, transferred to nitrocellulose and blotted with antibodies against human IgG (Thermo Scientific, cat.# 31413), human kappa LC (Thermo Scientific, cat.# 31414) or human lambda LC (Thermo Scientific, cat.# 31131).

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    Thermo Fisher tdp43 expression
    Effect of GluA3 antibodies in neurons differentiated from human iPSc on Tau and <t>TDP43</t> expression. ( A ) Representative WB analysis from homogenates of neurons differentiated from human iPSc and incubated for 24 hours with CSF with (CSF+) or without (CSF−) anti-GluA3 antibodies (dilution 1:100). ( B , C ) The histogram shows the quantification of the expression levels of Tau ( B ) and TDP43 ( C ), normalized on GAPDH; results are expressed as mean±standard errors (SEM), *p
    Tdp43 Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
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    88
    Thermo Fisher mir 29a expression
    Overexpression of <t>miR-29a</t> reduces weight gain, but has no effect on physical activity in the context of chronic high fat diet (HFD). Weight gain and physical activity of wild type and miR-29aTg mice fed a chow or high-fat diet for 12 months were measured, including ( A ) body weight, ( B ) frequency rearing stand, and ( C ) moving distance documented using a 30 × 30 cm open field box in ten minutes. Data calculated from five to ten mice per group are expressed as mean ± SE. ** p
    Mir 29a Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 3 article reviews
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    93
    Thermo Fisher live cell imaging cells expressing β tubulin gfp
    Loss of MYO5B causes cytokinesis delay. (A) Caco2 WT and Caco2 MYO5B−/− cells were fixed and stained as indicated. The percentage of cytokinesis with both daughter cells was quantified. n > 1,000 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data . (B) Live cell imaging shows x-y-t time-lapse mitosis images on Caco2 WT and Caco2 MYO5B−/− cells expressing <t>β-tubulin-GFP</t> and histone2B-mCherry. (C) The time of cytokinesis duration was quantified. (Left side graph) Each dot indicates 1 mitotic cell’s cytokinesis time. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 20 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data . t test, * p
    Live Cell Imaging Cells Expressing β Tubulin Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    93
    Thermo Fisher zp4 expression plasmids
    Mimicking the cumulus-oocyte complex (CB ZP C). ( a ) Expanded, native cumulus-oocyte complex (COC) after in vitro maturation. Scale bar, 30 μm (left). ZP-beads after 24 h of incubation with isolated cumulus cells obtained from in vitro matured COCs, cumulus-ZP coated beads complex (CB ZP C). Scale bar, 30 µm. The cells adhere to the bead surface resembling a native COC with two external coats, cumulus cells and ZP proteins (middle, right). ( b ) Quantification of the number of cells adhered to the ZP coated-bead (B ZP ) stained with 0.01 mM bisbenzimide and mechanically washed (mean ± SEM). No difference in the number of adherent cells was observed among the three zona proteins attached to sepharose beads (B ZP2, B ZP3 , B <t>ZP4</t> ). However, control beads without ZP proteins had fewer cells. Different letters ( a,b ) in the same column indicate differences between groups (P
    Zp4 Expression Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of GluA3 antibodies in neurons differentiated from human iPSc on Tau and TDP43 expression. ( A ) Representative WB analysis from homogenates of neurons differentiated from human iPSc and incubated for 24 hours with CSF with (CSF+) or without (CSF−) anti-GluA3 antibodies (dilution 1:100). ( B , C ) The histogram shows the quantification of the expression levels of Tau ( B ) and TDP43 ( C ), normalized on GAPDH; results are expressed as mean±standard errors (SEM), *p

    Journal: Scientific Reports

    Article Title: Anti-AMPA GluA3 antibodies in Frontotemporal dementia: a new molecular target

    doi: 10.1038/s41598-017-06117-y

    Figure Lengend Snippet: Effect of GluA3 antibodies in neurons differentiated from human iPSc on Tau and TDP43 expression. ( A ) Representative WB analysis from homogenates of neurons differentiated from human iPSc and incubated for 24 hours with CSF with (CSF+) or without (CSF−) anti-GluA3 antibodies (dilution 1:100). ( B , C ) The histogram shows the quantification of the expression levels of Tau ( B ) and TDP43 ( C ), normalized on GAPDH; results are expressed as mean±standard errors (SEM), *p

    Article Snippet: WB analysis was then performed in differentiated neuron total cell lysate to evaluate Tau and TDP43 expression, and unconjugated primary antibodies were used as follows: polyclonal Tau-5 antibody (Thermofisher) and polyclonal TDP43 antibody (Proteintech); for quantification, each protein was normalized against the corresponding GAPDH band (G9 antibody; Santa Cruz).

    Techniques: Expressing, Western Blot, Incubation

    Overexpression of miR-29a reduces weight gain, but has no effect on physical activity in the context of chronic high fat diet (HFD). Weight gain and physical activity of wild type and miR-29aTg mice fed a chow or high-fat diet for 12 months were measured, including ( A ) body weight, ( B ) frequency rearing stand, and ( C ) moving distance documented using a 30 × 30 cm open field box in ten minutes. Data calculated from five to ten mice per group are expressed as mean ± SE. ** p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: Overexpression of miR-29a reduces weight gain, but has no effect on physical activity in the context of chronic high fat diet (HFD). Weight gain and physical activity of wild type and miR-29aTg mice fed a chow or high-fat diet for 12 months were measured, including ( A ) body weight, ( B ) frequency rearing stand, and ( C ) moving distance documented using a 30 × 30 cm open field box in ten minutes. Data calculated from five to ten mice per group are expressed as mean ± SE. ** p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Over Expression, Activity Assay, Mouse Assay

    Overexpression of miR-29a represses hepatic epithelial-mesenchymal transition and inflammation in the context of chronic HFD. mRNA expression level of ( A ) vimentin, ( B ) snail, ( C ) mcp1, and ( D ) il6. β-actin level is used as the normalization control. Five to seven specimens were used for each group. Data are expressed as mean ± SE. * p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: Overexpression of miR-29a represses hepatic epithelial-mesenchymal transition and inflammation in the context of chronic HFD. mRNA expression level of ( A ) vimentin, ( B ) snail, ( C ) mcp1, and ( D ) il6. β-actin level is used as the normalization control. Five to seven specimens were used for each group. Data are expressed as mean ± SE. * p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Over Expression, Expressing

    Overexpression of miR-29a modulates HFD-caused perturbation of mitochondrial biogenesis in the liver. Representative immunoblotting bands and densitometric results of ( A ) peroxisome proliferator-activated receptor γ (PPARγ) and ( B ) mitochondrial transcription factor A (TFAM), using GAPDH as the loading control. ( C ) mtDNA copy number per cell probed using qPCR, with TERT as the normalization control. Five to ten specimens were used for each group. Data are expressed as mean ± SE. * p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: Overexpression of miR-29a modulates HFD-caused perturbation of mitochondrial biogenesis in the liver. Representative immunoblotting bands and densitometric results of ( A ) peroxisome proliferator-activated receptor γ (PPARγ) and ( B ) mitochondrial transcription factor A (TFAM), using GAPDH as the loading control. ( C ) mtDNA copy number per cell probed using qPCR, with TERT as the normalization control. Five to ten specimens were used for each group. Data are expressed as mean ± SE. * p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Over Expression, Real-time Polymerase Chain Reaction

    Overexpression of miR-29a reduces hepatocellular steatosis in the context of chronic HFD. Paraformaldehyde-fixed paraffin-embedded liver tissue was used to determine the abundance of lipid droplets with hematoxylin-eosin (HE) stain. ( A ) Representative HE stain image of each group. ( B ) Lipid droplet area quantified using ImageJ. Data collected from three fields of view of each specimen and six to nine specimens for each group are expressed as mean ± SE. ** p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: Overexpression of miR-29a reduces hepatocellular steatosis in the context of chronic HFD. Paraformaldehyde-fixed paraffin-embedded liver tissue was used to determine the abundance of lipid droplets with hematoxylin-eosin (HE) stain. ( A ) Representative HE stain image of each group. ( B ) Lipid droplet area quantified using ImageJ. Data collected from three fields of view of each specimen and six to nine specimens for each group are expressed as mean ± SE. ** p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Over Expression, H&E Stain

    The proposed model of miR-29a exerting a protective effect by targeting CD36 and modulating downstream signaling pathway in HFD-elicited liver fibrosis. HFD causes considerable fatty acid influx into the liver, leading to the up-regulation of PPARγ, TFAM, and mtDNA content. MtDNA and mitochondrial-derived reactive oxygen species can initiate an inflammatory response, leading to the release of such pro-inflammatory cytokines as MCP-1 and IL-6. Chronic inflammation is a stimulator for EMT, which is characterized by the up-regulation of typical makers like snail and vimentin. Activation of EMT facilitates the transformation of hepatic stellate cells to myofibroblasts, contributing to the progression of liver fibrosis. Of particular note, miR-29a can exert an anti-NAFLD effect by targeting CD36 3’UTR and repressing its expression, which may decrease intracellular fatty acid influx, subsequently reducing the up-regulation of PPARγ, TFAM, and mtDNA content, modulating downstream inflammatory response and EMT, and ultimately mitigating liver fibrosis.

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: The proposed model of miR-29a exerting a protective effect by targeting CD36 and modulating downstream signaling pathway in HFD-elicited liver fibrosis. HFD causes considerable fatty acid influx into the liver, leading to the up-regulation of PPARγ, TFAM, and mtDNA content. MtDNA and mitochondrial-derived reactive oxygen species can initiate an inflammatory response, leading to the release of such pro-inflammatory cytokines as MCP-1 and IL-6. Chronic inflammation is a stimulator for EMT, which is characterized by the up-regulation of typical makers like snail and vimentin. Activation of EMT facilitates the transformation of hepatic stellate cells to myofibroblasts, contributing to the progression of liver fibrosis. Of particular note, miR-29a can exert an anti-NAFLD effect by targeting CD36 3’UTR and repressing its expression, which may decrease intracellular fatty acid influx, subsequently reducing the up-regulation of PPARγ, TFAM, and mtDNA content, modulating downstream inflammatory response and EMT, and ultimately mitigating liver fibrosis.

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Derivative Assay, Activation Assay, Transformation Assay, Expressing

    MiR-29a inhibits the expression of fatty acid translocase CD36 by targeting 3’ untranslated region (UTR). ( A ) qPCR analysis of Cd36 in live tissue. ( B ) Representative immunoblotting bands and densitometric results of CD36 in liver tissue. ( C ) qPCR analysis of cd36 expression of HepG2 cells in vitro after 48h transfection of scramble sequence or miR-29a-mimic. Data obtained from six independent experiments. ( D ) Upper panel, sequence information, and mutual pairing status of CD36-3’UTR, mmu-miR29a, and CD36-3’UTR Mut. Note that red characters represent mismatching sites. HepG2 was first transfected with CD36-3’UTR or CD36-3’UTR mutant luciferase reporter construct then treated with control medium (ctrl), miRNA-scramble, or miR-29a mimic, and finally lysed to detect the luciferase signal. Data are expressed as mean ± SE. * p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: MiR-29a inhibits the expression of fatty acid translocase CD36 by targeting 3’ untranslated region (UTR). ( A ) qPCR analysis of Cd36 in live tissue. ( B ) Representative immunoblotting bands and densitometric results of CD36 in liver tissue. ( C ) qPCR analysis of cd36 expression of HepG2 cells in vitro after 48h transfection of scramble sequence or miR-29a-mimic. Data obtained from six independent experiments. ( D ) Upper panel, sequence information, and mutual pairing status of CD36-3’UTR, mmu-miR29a, and CD36-3’UTR Mut. Note that red characters represent mismatching sites. HepG2 was first transfected with CD36-3’UTR or CD36-3’UTR mutant luciferase reporter construct then treated with control medium (ctrl), miRNA-scramble, or miR-29a mimic, and finally lysed to detect the luciferase signal. Data are expressed as mean ± SE. * p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, In Vitro, Transfection, Sequencing, Mutagenesis, Luciferase, Construct

    Overexpression of miR-29a significantly reduces fat accumulation in adipose tissue and liver weight in the context of chronic HFD. Various tissue parts were dissected and weighed immediately after sacrifice, with weight of ( A ) subcutaneous, ( B ) visceral, ( C ) intestinal fat tissue, and ( D ) liver. Data calculated from seven to ten mice per group are expressed as mean ± SE. ** p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: Overexpression of miR-29a significantly reduces fat accumulation in adipose tissue and liver weight in the context of chronic HFD. Various tissue parts were dissected and weighed immediately after sacrifice, with weight of ( A ) subcutaneous, ( B ) visceral, ( C ) intestinal fat tissue, and ( D ) liver. Data calculated from seven to ten mice per group are expressed as mean ± SE. ** p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Over Expression, Mouse Assay

    Overexpression of miR-29a reduces liver fibrosis in the context of chronic HFD. Paraformaldehyde-fixed paraffin-embedded liver tissue was used to determine collagen fiber accumulation using Mason’s trichrome stain. Liver tissue stored at −80 ℃ was used for RNA and protein extraction for subsequent qPCR and Western blot experiments, respectively. ( A ) Representative Masson’s trichrome stain image of each group. Blue color indicates positive signal of collagen fiber. ( B ) Positive signal percentage quantified using ImageJ. ( C ) mRNA expression level of col1 α1 , with β-actin level as normalization control. ( D ) Representative immunoblotting bands of COL1α1 protein abundance and densitometric results, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the loading control. For the imaging study, data were collected from five fields of view of each specimen and five to eight specimens for each group. For qPCR and Western blot, five to seven specimens were used for each group. Data are expressed as mean ± SE. * p

    Journal: Cells

    Article Title: MicroRNA-29a Suppresses CD36 to Ameliorate High Fat Diet-Induced Steatohepatitis and Liver Fibrosis in Mice

    doi: 10.3390/cells8101298

    Figure Lengend Snippet: Overexpression of miR-29a reduces liver fibrosis in the context of chronic HFD. Paraformaldehyde-fixed paraffin-embedded liver tissue was used to determine collagen fiber accumulation using Mason’s trichrome stain. Liver tissue stored at −80 ℃ was used for RNA and protein extraction for subsequent qPCR and Western blot experiments, respectively. ( A ) Representative Masson’s trichrome stain image of each group. Blue color indicates positive signal of collagen fiber. ( B ) Positive signal percentage quantified using ImageJ. ( C ) mRNA expression level of col1 α1 , with β-actin level as normalization control. ( D ) Representative immunoblotting bands of COL1α1 protein abundance and densitometric results, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the loading control. For the imaging study, data were collected from five fields of view of each specimen and five to eight specimens for each group. For qPCR and Western blot, five to seven specimens were used for each group. Data are expressed as mean ± SE. * p

    Article Snippet: For detection of miR-29a expression, predesigned primer/probes for miR-29a (#002112, ThermoFisher) and normalization control sno202 (#001232, ThermoFisher) were used.

    Techniques: Over Expression, Staining, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Imaging

    Loss of MYO5B causes cytokinesis delay. (A) Caco2 WT and Caco2 MYO5B−/− cells were fixed and stained as indicated. The percentage of cytokinesis with both daughter cells was quantified. n > 1,000 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data . (B) Live cell imaging shows x-y-t time-lapse mitosis images on Caco2 WT and Caco2 MYO5B−/− cells expressing β-tubulin-GFP and histone2B-mCherry. (C) The time of cytokinesis duration was quantified. (Left side graph) Each dot indicates 1 mitotic cell’s cytokinesis time. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 20 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data . t test, * p

    Journal: PLoS Biology

    Article Title: Loss of MYO5B expression deregulates late endosome size which hinders mitotic spindle orientation

    doi: 10.1371/journal.pbio.3000531

    Figure Lengend Snippet: Loss of MYO5B causes cytokinesis delay. (A) Caco2 WT and Caco2 MYO5B−/− cells were fixed and stained as indicated. The percentage of cytokinesis with both daughter cells was quantified. n > 1,000 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data . (B) Live cell imaging shows x-y-t time-lapse mitosis images on Caco2 WT and Caco2 MYO5B−/− cells expressing β-tubulin-GFP and histone2B-mCherry. (C) The time of cytokinesis duration was quantified. (Left side graph) Each dot indicates 1 mitotic cell’s cytokinesis time. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 20 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data . t test, * p

    Article Snippet: Live cell imaging Cells expressing β-tubulin-GFP and histone2B-mCherry were seeded in an 8-chambers plate (Lab-Tek II, 155409) ( Thermo Scientific , Waltham, MA, USA) and incubated with DMEM medium for 72 h. Then the live cells were observed 24 h with the laser scanning confocal microscope (GE Healthcare Bio-Sciences, Marlborough, MA, USA) with temperature and CO2 control.

    Techniques: Staining, Live Cell Imaging, Expressing

    Mitotic spindle orientation defects correlated to the presence of large vacuoles in MYO5B-depleted cells. (A) Live cell imaging shows mitosis images on Caco2 MYO5B−/− cells expressing β-tubulin-GFP and histone2B-mCherry. The α angle represents the angle between a line drawn through the spindle axis at the onset of metaphase (dotted line) and the end of metaphase (solid line). Dotted circles indicate large vacuoles which were confirmed by bright field. (B) The α angle in Caco2 MYO5B−/− cells with vacuole and without vacuole during metaphase was quantified. Each dot indicating 1 mitotic cell’s spindle rotation α angle (left). The statistical analysis of the mean for each experiment (right). n > 15 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S4 Data . (C) The position of mitotic spindle relative to vacuole during onset and end of metaphase in Caco2 MYO5B−/− cells was quantified by measuring the distance from spindle pole to vacuole (d, red line). Arrows in the bright field and dotted circles in the fluorescent field indicate large vacuoles. (D) The comparison of the distance d in the onset and end of metaphase in Caco2 MYO5B−/− cells. Values for each data point can be found in S4 Data . (E) The quantification of the distance d in vacuolated Caco2 MYO5B−/− cells. (Left side graph) Each dot indicates one cell’s distance d between the onset and end of metaphase. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 5 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S4 Data . t test, ** p

    Journal: PLoS Biology

    Article Title: Loss of MYO5B expression deregulates late endosome size which hinders mitotic spindle orientation

    doi: 10.1371/journal.pbio.3000531

    Figure Lengend Snippet: Mitotic spindle orientation defects correlated to the presence of large vacuoles in MYO5B-depleted cells. (A) Live cell imaging shows mitosis images on Caco2 MYO5B−/− cells expressing β-tubulin-GFP and histone2B-mCherry. The α angle represents the angle between a line drawn through the spindle axis at the onset of metaphase (dotted line) and the end of metaphase (solid line). Dotted circles indicate large vacuoles which were confirmed by bright field. (B) The α angle in Caco2 MYO5B−/− cells with vacuole and without vacuole during metaphase was quantified. Each dot indicating 1 mitotic cell’s spindle rotation α angle (left). The statistical analysis of the mean for each experiment (right). n > 15 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S4 Data . (C) The position of mitotic spindle relative to vacuole during onset and end of metaphase in Caco2 MYO5B−/− cells was quantified by measuring the distance from spindle pole to vacuole (d, red line). Arrows in the bright field and dotted circles in the fluorescent field indicate large vacuoles. (D) The comparison of the distance d in the onset and end of metaphase in Caco2 MYO5B−/− cells. Values for each data point can be found in S4 Data . (E) The quantification of the distance d in vacuolated Caco2 MYO5B−/− cells. (Left side graph) Each dot indicates one cell’s distance d between the onset and end of metaphase. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 5 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S4 Data . t test, ** p

    Article Snippet: Live cell imaging Cells expressing β-tubulin-GFP and histone2B-mCherry were seeded in an 8-chambers plate (Lab-Tek II, 155409) ( Thermo Scientific , Waltham, MA, USA) and incubated with DMEM medium for 72 h. Then the live cells were observed 24 h with the laser scanning confocal microscope (GE Healthcare Bio-Sciences, Marlborough, MA, USA) with temperature and CO2 control.

    Techniques: Live Cell Imaging, Expressing

    Mimicking the cumulus-oocyte complex (CB ZP C). ( a ) Expanded, native cumulus-oocyte complex (COC) after in vitro maturation. Scale bar, 30 μm (left). ZP-beads after 24 h of incubation with isolated cumulus cells obtained from in vitro matured COCs, cumulus-ZP coated beads complex (CB ZP C). Scale bar, 30 µm. The cells adhere to the bead surface resembling a native COC with two external coats, cumulus cells and ZP proteins (middle, right). ( b ) Quantification of the number of cells adhered to the ZP coated-bead (B ZP ) stained with 0.01 mM bisbenzimide and mechanically washed (mean ± SEM). No difference in the number of adherent cells was observed among the three zona proteins attached to sepharose beads (B ZP2, B ZP3 , B ZP4 ). However, control beads without ZP proteins had fewer cells. Different letters ( a,b ) in the same column indicate differences between groups (P

    Journal: Scientific Reports

    Article Title: Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

    doi: 10.1038/s41598-019-54501-7

    Figure Lengend Snippet: Mimicking the cumulus-oocyte complex (CB ZP C). ( a ) Expanded, native cumulus-oocyte complex (COC) after in vitro maturation. Scale bar, 30 μm (left). ZP-beads after 24 h of incubation with isolated cumulus cells obtained from in vitro matured COCs, cumulus-ZP coated beads complex (CB ZP C). Scale bar, 30 µm. The cells adhere to the bead surface resembling a native COC with two external coats, cumulus cells and ZP proteins (middle, right). ( b ) Quantification of the number of cells adhered to the ZP coated-bead (B ZP ) stained with 0.01 mM bisbenzimide and mechanically washed (mean ± SEM). No difference in the number of adherent cells was observed among the three zona proteins attached to sepharose beads (B ZP2, B ZP3 , B ZP4 ). However, control beads without ZP proteins had fewer cells. Different letters ( a,b ) in the same column indicate differences between groups (P

    Article Snippet: After verification by DNA sequence, ZP2C, ZP2N, ZP3 and ZP4 expression plasmids were amplified using Library Efficiency DH5α Competent cells (Thermo Fisher Scientific) and purified with GenEluted Plasmid Kit.

    Techniques: In Vitro, Incubation, Isolation, Staining

    Conjugation of ZP recombinant proteins to sepharose magnetic beads. ( a ) Schematic representation of ZP recombinant proteins coated beads (B ZP2, B ZP3 and B ZP4 ) (upper). SDS-PAGE and western blot of ZP2C, ZP3 and ZP4 proteins conjugated to magnetic beads (lower). Medium with secreted proteins before conjugation (lane 1), in the eluted fraction (lane 2), and media after conjugation (lane 3). Anti-Flag antibody was used for ZP2, anti-ZP3 for ZP3 and V5 Epitope Tag antibody for ZP4. ( b ) Confocal microscopy images of beads alone (B Ctrl ) (left) and conjugated beads (B ZP ) (right) showing uniform coating of beads with ZP proteins. Scale bar, 40 μm. ( c) SDS-PAGE and western blot of ZP2C, ZP3 and ZP4 proteins conjugated to magnetic beads after conjugation and storage for 0, 24, 48, 72 and 144 h. Lane C, beads incubated with CHO-cell growth medium without recombinant zona proteins.

    Journal: Scientific Reports

    Article Title: Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

    doi: 10.1038/s41598-019-54501-7

    Figure Lengend Snippet: Conjugation of ZP recombinant proteins to sepharose magnetic beads. ( a ) Schematic representation of ZP recombinant proteins coated beads (B ZP2, B ZP3 and B ZP4 ) (upper). SDS-PAGE and western blot of ZP2C, ZP3 and ZP4 proteins conjugated to magnetic beads (lower). Medium with secreted proteins before conjugation (lane 1), in the eluted fraction (lane 2), and media after conjugation (lane 3). Anti-Flag antibody was used for ZP2, anti-ZP3 for ZP3 and V5 Epitope Tag antibody for ZP4. ( b ) Confocal microscopy images of beads alone (B Ctrl ) (left) and conjugated beads (B ZP ) (right) showing uniform coating of beads with ZP proteins. Scale bar, 40 μm. ( c) SDS-PAGE and western blot of ZP2C, ZP3 and ZP4 proteins conjugated to magnetic beads after conjugation and storage for 0, 24, 48, 72 and 144 h. Lane C, beads incubated with CHO-cell growth medium without recombinant zona proteins.

    Article Snippet: After verification by DNA sequence, ZP2C, ZP2N, ZP3 and ZP4 expression plasmids were amplified using Library Efficiency DH5α Competent cells (Thermo Fisher Scientific) and purified with GenEluted Plasmid Kit.

    Techniques: Conjugation Assay, Recombinant, Magnetic Beads, SDS Page, Western Blot, Confocal Microscopy, Incubation

    Design and expression of porcine recombinant ZP proteins. (a ) Schematic representation of recombinant porcine ZP glycoproteins, ZP2C, ZP3 and ZP4. Signal peptide (pink), ZP domain (blue), processing region (green) and transmembrane domain (orange). ( b) Proteins were expressed in CHO cells, separated by SDS-PAGE and analysed by western blot. ZP proteins were probed with anti-Flag antibodies for ZP2C, anti-ZP3 for ZP3 and V5 Epitope Tag antibody for ZP4. Molecular mass markers, left.

    Journal: Scientific Reports

    Article Title: Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

    doi: 10.1038/s41598-019-54501-7

    Figure Lengend Snippet: Design and expression of porcine recombinant ZP proteins. (a ) Schematic representation of recombinant porcine ZP glycoproteins, ZP2C, ZP3 and ZP4. Signal peptide (pink), ZP domain (blue), processing region (green) and transmembrane domain (orange). ( b) Proteins were expressed in CHO cells, separated by SDS-PAGE and analysed by western blot. ZP proteins were probed with anti-Flag antibodies for ZP2C, anti-ZP3 for ZP3 and V5 Epitope Tag antibody for ZP4. Molecular mass markers, left.

    Article Snippet: After verification by DNA sequence, ZP2C, ZP2N, ZP3 and ZP4 expression plasmids were amplified using Library Efficiency DH5α Competent cells (Thermo Fisher Scientific) and purified with GenEluted Plasmid Kit.

    Techniques: Expressing, Recombinant, SDS Page, Western Blot