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Millipore ripa lysis buffer
Ripa Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
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ripa lysis buffer - by Bioz Stars, 2021-03
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Incubation:

Article Title: Host Cell Cathepsins Potentiate Moloney Murine Leukemia Virus Infection ▿
Article Snippet: .. For analysis of cathepsin B cleavage, aliquots of pelleted virus were incubated at 37°C for 1 h in acetate buffer (pH 5.5) plus or minus the specified amount of cathepsin B (Calbiochem). .. In some cases, 100 μM CA-074 was added along with the cathepsin B, and in some cases, reaction mixtures were incubated for longer periods, up to 4 h at 37°C.

Stable Transfection:

Article Title: Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA
Article Snippet: .. For anti-FLAG IP under native conditions, HEK293 cells stably expressing FLAG-tagged EJC protein were lysed in ice-cold hypotonic lysis buffer (HLB) [20 mM Tris-HCl pH 7.5, 15 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, 1× Sigma protease inhibitor cocktail, 1 mM PMSF]. .. Lysates were sonicated, supplemented with NaCl to a final concentration of 150 mM and RNase A was added to 125 µg/mL.

Expressing:

Article Title: Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA
Article Snippet: .. For anti-FLAG IP under native conditions, HEK293 cells stably expressing FLAG-tagged EJC protein were lysed in ice-cold hypotonic lysis buffer (HLB) [20 mM Tris-HCl pH 7.5, 15 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, 1× Sigma protease inhibitor cocktail, 1 mM PMSF]. .. Lysates were sonicated, supplemented with NaCl to a final concentration of 150 mM and RNase A was added to 125 µg/mL.

Lysis:

Article Title: Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA
Article Snippet: .. For anti-FLAG IP under native conditions, HEK293 cells stably expressing FLAG-tagged EJC protein were lysed in ice-cold hypotonic lysis buffer (HLB) [20 mM Tris-HCl pH 7.5, 15 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, 1× Sigma protease inhibitor cocktail, 1 mM PMSF]. .. Lysates were sonicated, supplemented with NaCl to a final concentration of 150 mM and RNase A was added to 125 µg/mL.

Protease Inhibitor:

Article Title: Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA
Article Snippet: .. For anti-FLAG IP under native conditions, HEK293 cells stably expressing FLAG-tagged EJC protein were lysed in ice-cold hypotonic lysis buffer (HLB) [20 mM Tris-HCl pH 7.5, 15 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, 1× Sigma protease inhibitor cocktail, 1 mM PMSF]. .. Lysates were sonicated, supplemented with NaCl to a final concentration of 150 mM and RNase A was added to 125 µg/mL.

Western Blot:

Article Title: Protein Kinase D1: A New Component in Toll-Like Receptor 9 Signaling
Article Snippet: RAW264.7 cells stably expressing empty vector, FLAG-tagged PKD1, FLAG-tagged PKD2, or FLAG-tagged PKD3 were stimulated with medium or CpG-B DNA for 30 min. Each FLAG-tagged PKD family protein or endogenous protein in TLR9 signaling (TLR9, MyD88, IRAK1, IRAK4, TRAF6, or TRAF3) in the cell lysates was immunoprecipitated with EZview Red ANTI-FLAG M2 Affinity Gel (Sigma) or Abs specific for each individual protein. .. The presence of PKDs and proteins in the TLR9-signaling pathway in the resulting immunoprecipitates was detected by Western blot using Abs specific for FLAG (Sigma), TLR9 (Imgenex, San Diego, CA), IRAK1 (Millipore, Billerica, MA), IRAK4 (Abgent, San Diego, CA), MyD88 (Santa Cruz), TRAF6 (Santa Cruz), or TRAF3 (Santa Cruz). .. PKD family proteins are expressed in T- and B-lymphocytes and play a regulatory role in TCR- and BCR-mediated lymphocyte activation ( , ).

Recombinant:

Article Title: PKR and GCN2 Kinases and Guanine Nucleotide Exchange Factor Eukaryotic Translation Initiation Factor 2B (eIF2B) Recognize Overlapping Surfaces on eIF2?
Article Snippet: Because the sequences of the six plasmids from the residue-89-to-288 screen revealed multiple mutations and Western blot analyses using phosphospecific antibodies against Ser51 indicated that the mutations did not interfere with eIF2α phosphorylation, these mutants were not examined further. .. GST-eIF2α fusion proteins were expressed in Escherichia coli BL21(DE3) plus cells (Novagen), and the recombinant proteins were purified as previously described ( ). ..

Purification:

Article Title: PKR and GCN2 Kinases and Guanine Nucleotide Exchange Factor Eukaryotic Translation Initiation Factor 2B (eIF2B) Recognize Overlapping Surfaces on eIF2?
Article Snippet: Because the sequences of the six plasmids from the residue-89-to-288 screen revealed multiple mutations and Western blot analyses using phosphospecific antibodies against Ser51 indicated that the mutations did not interfere with eIF2α phosphorylation, these mutants were not examined further. .. GST-eIF2α fusion proteins were expressed in Escherichia coli BL21(DE3) plus cells (Novagen), and the recombinant proteins were purified as previously described ( ). ..

Double Immunofluorescence Staining:

Article Title: Ultrastructural and immunohistochemical study of the basal apparatus of solitary cilia in the human oviduct epithelium
Article Snippet: .. Double immunofluorescence staining was carried out as described previously ( ) using a mouse monoclonal antibody against γ-tubulin (Sigma, St Louis, MO, USA), a rat monoclonal antibody R67 against the proteins of 205/215 kDa of striated rootlets , FITC-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA, USA) and rhodamine-conjugated goat anti-rat IgG (Chemicon). .. The sections were examined with an Axioplan fluorescence microscope (Carl Zeiss, Tokyo, Japan) and/or an MRC-1024ES confocal laser scanning microscope (Bio-Rad, Tokyo, Japan).

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    Millipore ripa buffer
    Differential induction of Peli1 and TRAF3 degradation in TLR3- and TLR4-stimulated bid-deficient glia. a , b wt and bid −/− macrophages were stimulated with PolyI:C (100 ng/ml) for 1 h, lysed in <t>RIPA</t> buffer, and prepared for Western blot where Peli1 levels were determined ( n = 5, 5 cultures from 4 separate platings, p = 0.004 one-way ANOVA, Tukey’s post hoc test). c , d wt and bid -deficient astrocytes were stimulated with <t>LPS</t> (100 ng/ml) for 5, 15, and 30 min and 1 h before being lysed in RIPA buffer and prepared for Western blot analysis of Peli1 levels ( n = 4–5, 4–5 cultures from 3 separate platings, p = 0.031, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test). e , f wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and TRAF3 levels were determined by Western blot ( n = 3–4, 3–4 cultures from 4 separate platings, p = 0.019 one-way ANOVA, Tukey’s multiple comparison post hoc test). All samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein levels were quantified using optical densities. The data are represented as mean ± SD
    Ripa Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ripa buffer - by Bioz Stars, 2021-03
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    Differential induction of Peli1 and TRAF3 degradation in TLR3- and TLR4-stimulated bid-deficient glia. a , b wt and bid −/− macrophages were stimulated with PolyI:C (100 ng/ml) for 1 h, lysed in RIPA buffer, and prepared for Western blot where Peli1 levels were determined ( n = 5, 5 cultures from 4 separate platings, p = 0.004 one-way ANOVA, Tukey’s post hoc test). c , d wt and bid -deficient astrocytes were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min and 1 h before being lysed in RIPA buffer and prepared for Western blot analysis of Peli1 levels ( n = 4–5, 4–5 cultures from 3 separate platings, p = 0.031, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test). e , f wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and TRAF3 levels were determined by Western blot ( n = 3–4, 3–4 cultures from 4 separate platings, p = 0.019 one-way ANOVA, Tukey’s multiple comparison post hoc test). All samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein levels were quantified using optical densities. The data are represented as mean ± SD

    Journal: Journal of Neuroinflammation

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

    doi: 10.1186/s12974-018-1143-3

    Figure Lengend Snippet: Differential induction of Peli1 and TRAF3 degradation in TLR3- and TLR4-stimulated bid-deficient glia. a , b wt and bid −/− macrophages were stimulated with PolyI:C (100 ng/ml) for 1 h, lysed in RIPA buffer, and prepared for Western blot where Peli1 levels were determined ( n = 5, 5 cultures from 4 separate platings, p = 0.004 one-way ANOVA, Tukey’s post hoc test). c , d wt and bid -deficient astrocytes were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min and 1 h before being lysed in RIPA buffer and prepared for Western blot analysis of Peli1 levels ( n = 4–5, 4–5 cultures from 3 separate platings, p = 0.031, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test). e , f wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and TRAF3 levels were determined by Western blot ( n = 3–4, 3–4 cultures from 4 separate platings, p = 0.019 one-way ANOVA, Tukey’s multiple comparison post hoc test). All samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein levels were quantified using optical densities. The data are represented as mean ± SD

    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma).

    Techniques: Western Blot

    Bid deficiency does not alter autophagosome formation . a , b wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 30 min or 1 h and lysed in RIPA buffer, and anti-LC3-II levels were determined by Western blot ( n = 3, cultures from 3 separate platings, one-way ANOVA, p = 0.43). The data are represented as mean ± SD

    Journal: Journal of Neuroinflammation

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

    doi: 10.1186/s12974-018-1143-3

    Figure Lengend Snippet: Bid deficiency does not alter autophagosome formation . a , b wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 30 min or 1 h and lysed in RIPA buffer, and anti-LC3-II levels were determined by Western blot ( n = 3, cultures from 3 separate platings, one-way ANOVA, p = 0.43). The data are represented as mean ± SD

    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma).

    Techniques: Western Blot

    Bid-dependent induction of Peli1 in TLR3- and TLR4-activated microglia . a wt and bid -deficient microglia were stimulated with PolyI:C (100 ng/ml) for 1 h, lysed in RLT buffer, and prepared for qPCR analysis where Peli1 mRNA was quantified using the LightCycler ( n = 6–7, 6–7 cultures from 6 separate platings, p = 0.0143, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test) . b wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h, lysed in RLT buffer, and prepared for qPCR analysis where Peli1 mRNA was quantified using the LightCycler ( n = 5–7, 5–7 cultures from 3 separate platings, p = 0.0025 one-way ANOVA, Tukey’s multiple comparison post hoc test). c , d wt and bid -deficient microglia were treated with Bortezomib (100 μM) for 1, 2, or 6 h, before being lysed in RIPA buffer, and Peli1 levels were determined by Western blot ( n = 3–4, 3–4 cultures from 4 separate platings, p = 0.072, one-way ANOVA). e , f wt and bid -deficient microglia were treated with Bortezomib (100 μM) for 1, 2, or 6 h, before being lysed in RIPA buffer, and TRAF3 levels were determined by Western blot ( n = 3, 3 cultures from 3 separate platings, p = 0.123, one-way ANOVA). All samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein levels were quantified using optical densities. The data are represented as mean ± SD

    Journal: Journal of Neuroinflammation

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

    doi: 10.1186/s12974-018-1143-3

    Figure Lengend Snippet: Bid-dependent induction of Peli1 in TLR3- and TLR4-activated microglia . a wt and bid -deficient microglia were stimulated with PolyI:C (100 ng/ml) for 1 h, lysed in RLT buffer, and prepared for qPCR analysis where Peli1 mRNA was quantified using the LightCycler ( n = 6–7, 6–7 cultures from 6 separate platings, p = 0.0143, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test) . b wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h, lysed in RLT buffer, and prepared for qPCR analysis where Peli1 mRNA was quantified using the LightCycler ( n = 5–7, 5–7 cultures from 3 separate platings, p = 0.0025 one-way ANOVA, Tukey’s multiple comparison post hoc test). c , d wt and bid -deficient microglia were treated with Bortezomib (100 μM) for 1, 2, or 6 h, before being lysed in RIPA buffer, and Peli1 levels were determined by Western blot ( n = 3–4, 3–4 cultures from 4 separate platings, p = 0.072, one-way ANOVA). e , f wt and bid -deficient microglia were treated with Bortezomib (100 μM) for 1, 2, or 6 h, before being lysed in RIPA buffer, and TRAF3 levels were determined by Western blot ( n = 3, 3 cultures from 3 separate platings, p = 0.123, one-way ANOVA). All samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein levels were quantified using optical densities. The data are represented as mean ± SD

    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma).

    Techniques: Real-time Polymerase Chain Reaction, Western Blot

    Reduced phosphorylation of TBK1, IRF3, and c-Jun in bid-deficient glia and macrophages compared with wt. a , b wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and phosphorylated TBK1 levels were determined by Western blot ( n = 6–7, 6–7 cultures from 6 separate platings, p = 0.0068, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test). c , d wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min or 1 h and lysed in RIPA buffer, and phosphorylated-IRF3 levels were determined by Western blot ( n = 3, 3 cultures from 3 separate platings, p = 0.041, paired two-tailed t test, wt Veh vs. LPS 1 h). e , f wt and bid −/− macrophages were stimulated with PolyI:C (100 ng/ml) for 30 min or 1 h and lysed in RIPA buffer, and phosphorylated-TBK1 levels were determined by Western blot ( n = 4–6, 4–6 cultures from 5 separate platings, p = 0.0006 one-way ANOVA, Tukey’s multiple comparison post hoc test). g , h wt macrophages were transfected with an siRNA targeting Bid. Forty-eight hours post transfection, when Bid is optimally silenced, the cells were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and phosphorylated-TBK1 levels were determined by Western blot ( n = 3, 3 cultures from 2 separate platings, p = 0.0588, paired two-tailed t test siControl LPS 1 h vs. siBid LPS 1 h). i , j wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min or 1 h and lysed in RIPA buffer, and phosphorylated c-Jun levels were determined by Western blot ( n = 5–7, 5–7 cultures from 6 separate platings, p

    Journal: Journal of Neuroinflammation

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

    doi: 10.1186/s12974-018-1143-3

    Figure Lengend Snippet: Reduced phosphorylation of TBK1, IRF3, and c-Jun in bid-deficient glia and macrophages compared with wt. a , b wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and phosphorylated TBK1 levels were determined by Western blot ( n = 6–7, 6–7 cultures from 6 separate platings, p = 0.0068, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test). c , d wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min or 1 h and lysed in RIPA buffer, and phosphorylated-IRF3 levels were determined by Western blot ( n = 3, 3 cultures from 3 separate platings, p = 0.041, paired two-tailed t test, wt Veh vs. LPS 1 h). e , f wt and bid −/− macrophages were stimulated with PolyI:C (100 ng/ml) for 30 min or 1 h and lysed in RIPA buffer, and phosphorylated-TBK1 levels were determined by Western blot ( n = 4–6, 4–6 cultures from 5 separate platings, p = 0.0006 one-way ANOVA, Tukey’s multiple comparison post hoc test). g , h wt macrophages were transfected with an siRNA targeting Bid. Forty-eight hours post transfection, when Bid is optimally silenced, the cells were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and phosphorylated-TBK1 levels were determined by Western blot ( n = 3, 3 cultures from 2 separate platings, p = 0.0588, paired two-tailed t test siControl LPS 1 h vs. siBid LPS 1 h). i , j wt and bid -deficient microglia were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min or 1 h and lysed in RIPA buffer, and phosphorylated c-Jun levels were determined by Western blot ( n = 5–7, 5–7 cultures from 6 separate platings, p

    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma).

    Techniques: Western Blot, Two Tailed Test, Transfection

    Increased A20-TRAF6, A20-TRAF3, and Smad6-A20 interactions in bid-deficient glia. a wt and bid −/− mixed glia were transfected with TRAF6-FLAG and stimulated for 1 h with LPS (100 ng/ml) 20 h post transfection. The cells were lysed in RIPA buffer, and anti-FLAG was immunoprecipitated from the samples. A20-TRAF6-FLAG interaction was detected by Western blotting using an anti-A20 antibody ( n = 2 separate experiments, repeated with similar results). b wt and bid −/− glia were transfected with ubiquitin-HA for 20 h and subsequently stimulated with Pam 3 CSK4 (Pam, 100 ng/ml) or PolyI:C (100 ng/ml) for 1 h before being lysed in RIPA buffer. Anti-TRAF3 was co-immunoprecipitated from each of the samples, and TRAF3-A20 interactions were determined by Western blotting using anti-A20 ( n = 1 experiment). c wt and bid -deficient mixed glia were transfected with Smad6-FLAG, or control vector pcDNA4.1, 20 h prior to stimulation with PolyI:C (100 ng/ml) or LPS (100 ng/ml) in full serum media. Following a 1-h stimulation, the cells were lysed in RIPA buffer, containing protease and phosphatase inhibitors, and the protein concentration was assessed using BCA assay. Anti-FLAG was immunoprecipitated from each of the samples, and the samples were prepared for Western blot analysis. The membrane was exposed to anti-A20 to determine the Smad6-FLAG-A20 interactions ( n = 2 experiments, repeated with similar results). All samples were lysed in RIPA buffer containing protease inhibitors, and protein concentrations were determined by BCA assay. Anti-IgG was used as co-immunoprecipitation and pull-down controls for each experiment. d Schematic showing the mechanism of action of Bid in response to TLR2 and − 3 and − 4 stimulation in glia and macrophages. Bid sequesters Smad6 and A20 thereby preventing Smad6-mediated recruitment of A20 to the E3 ligases, resulting in a lack of polyubiquitin chain cleavage and promotes pro-inflammatory signaling

    Journal: Journal of Neuroinflammation

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

    doi: 10.1186/s12974-018-1143-3

    Figure Lengend Snippet: Increased A20-TRAF6, A20-TRAF3, and Smad6-A20 interactions in bid-deficient glia. a wt and bid −/− mixed glia were transfected with TRAF6-FLAG and stimulated for 1 h with LPS (100 ng/ml) 20 h post transfection. The cells were lysed in RIPA buffer, and anti-FLAG was immunoprecipitated from the samples. A20-TRAF6-FLAG interaction was detected by Western blotting using an anti-A20 antibody ( n = 2 separate experiments, repeated with similar results). b wt and bid −/− glia were transfected with ubiquitin-HA for 20 h and subsequently stimulated with Pam 3 CSK4 (Pam, 100 ng/ml) or PolyI:C (100 ng/ml) for 1 h before being lysed in RIPA buffer. Anti-TRAF3 was co-immunoprecipitated from each of the samples, and TRAF3-A20 interactions were determined by Western blotting using anti-A20 ( n = 1 experiment). c wt and bid -deficient mixed glia were transfected with Smad6-FLAG, or control vector pcDNA4.1, 20 h prior to stimulation with PolyI:C (100 ng/ml) or LPS (100 ng/ml) in full serum media. Following a 1-h stimulation, the cells were lysed in RIPA buffer, containing protease and phosphatase inhibitors, and the protein concentration was assessed using BCA assay. Anti-FLAG was immunoprecipitated from each of the samples, and the samples were prepared for Western blot analysis. The membrane was exposed to anti-A20 to determine the Smad6-FLAG-A20 interactions ( n = 2 experiments, repeated with similar results). All samples were lysed in RIPA buffer containing protease inhibitors, and protein concentrations were determined by BCA assay. Anti-IgG was used as co-immunoprecipitation and pull-down controls for each experiment. d Schematic showing the mechanism of action of Bid in response to TLR2 and − 3 and − 4 stimulation in glia and macrophages. Bid sequesters Smad6 and A20 thereby preventing Smad6-mediated recruitment of A20 to the E3 ligases, resulting in a lack of polyubiquitin chain cleavage and promotes pro-inflammatory signaling

    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Protein Concentration, BIA-KA

    Differential expression of CAIX and CAXII in patient-derived tumor grafts. Panel A. Frozen tissue samples from PDX tumors were homogenized in RIPA buffer containing protease inhibitor. Western blots were probed with antibodies for CAIX, CAXII, CAII, ER, E-cadherin, actin, and GAPDH. Panel B. Tumor growth rates of orthotopically implanted, cryo-preserved tumor tissue was evaluated in NOD/SCID mice. Panel C. Immunohistochemistry was utilized to evaluate expression of CAIX, CAXII, ER, and Ki67 in TNBC (HCl-001) and ER/PR-positive (HCl-011) tumors from Panel B. Magnification: primary objective magnification, 10x.

    Journal: PLoS ONE

    Article Title: Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

    doi: 10.1371/journal.pone.0199476

    Figure Lengend Snippet: Differential expression of CAIX and CAXII in patient-derived tumor grafts. Panel A. Frozen tissue samples from PDX tumors were homogenized in RIPA buffer containing protease inhibitor. Western blots were probed with antibodies for CAIX, CAXII, CAII, ER, E-cadherin, actin, and GAPDH. Panel B. Tumor growth rates of orthotopically implanted, cryo-preserved tumor tissue was evaluated in NOD/SCID mice. Panel C. Immunohistochemistry was utilized to evaluate expression of CAIX, CAXII, ER, and Ki67 in TNBC (HCl-001) and ER/PR-positive (HCl-011) tumors from Panel B. Magnification: primary objective magnification, 10x.

    Article Snippet: Frozen PDX samples were homogenized in RIPA buffer containing protease inhibitor (Sigma # P8340) using an Omni homogenizer.

    Techniques: Expressing, Derivative Assay, Protease Inhibitor, Western Blot, Mouse Assay, Immunohistochemistry

    Characterization of cardiomyocyte-derived extracellular vesicles. (A) Representative electron microscopy images of isolated extracellular vesicles (EVs) collected from cardiomyocyte (CM) cultures in normoxia (Nx) or hypoxia (Hx) ( n = 3). Scale bars = 200 nm. (B) Representative western blot of common proteins found in EVs. EVs were lysed in RIPA buffer with complete protease inhibitors. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. (C) Representative images of EVs analyzed on the NanoSight NS300 instrument: particles/mL on vertical axis and size in nanometers (nm) on horizontal axis ( n = 5). (D) Protein quantification of EVs harvested from equal amounts of conditioned medium ( n = 3, ** p

    Journal: Frontiers in Cardiovascular Medicine

    Article Title: Extracellular Vesicles Secreted by Hypoxic AC10 Cardiomyocytes Modulate Fibroblast Cell Motility

    doi: 10.3389/fcvm.2018.00152

    Figure Lengend Snippet: Characterization of cardiomyocyte-derived extracellular vesicles. (A) Representative electron microscopy images of isolated extracellular vesicles (EVs) collected from cardiomyocyte (CM) cultures in normoxia (Nx) or hypoxia (Hx) ( n = 3). Scale bars = 200 nm. (B) Representative western blot of common proteins found in EVs. EVs were lysed in RIPA buffer with complete protease inhibitors. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. (C) Representative images of EVs analyzed on the NanoSight NS300 instrument: particles/mL on vertical axis and size in nanometers (nm) on horizontal axis ( n = 5). (D) Protein quantification of EVs harvested from equal amounts of conditioned medium ( n = 3, ** p

    Article Snippet: EVs were suspended in RIPA buffer [1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate in Tris-buffered saline (TBS), Sigma-Aldrich] for western blotting or phosphate buffered saline (PBS) for nanoparticle tracking, electron microscopy, flow cytometry, proteomic and functional analysis.

    Techniques: Derivative Assay, Electron Microscopy, Isolation, Western Blot

    GR-dependent up-regulation of CBR1 transcription by cortisol. A, siRNA-mediated knockdown of GR attenuated cortisol-induced CBR1 mRNA expression. B, Chromatin immunoprecipitation (ChIP) assay revealed increased enrichment of GR and RNA polymerase II (Pol

    Journal: Endocrinology

    Article Title: Induction of PGF2α Synthesis by Cortisol Through GR Dependent Induction of CBR1 in Human Amnion Fibroblasts

    doi: 10.1210/en.2013-1848

    Figure Lengend Snippet: GR-dependent up-regulation of CBR1 transcription by cortisol. A, siRNA-mediated knockdown of GR attenuated cortisol-induced CBR1 mRNA expression. B, Chromatin immunoprecipitation (ChIP) assay revealed increased enrichment of GR and RNA polymerase II (Pol

    Article Snippet: Total RNA and protein were extracted from homogenized amnion tissue or cultured amnion fibroblast cells using a RNeasy mini extraction kit (QIAGEN) and radio-immunoprecipitation assay lysis buffer (Millipore) respectively.

    Techniques: Expressing, Chromatin Immunoprecipitation