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  • 99
    Vector Laboratories anti rabbit antibody
    Anti Rabbit Antibody, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Biotium anti rabbit ab
    Anti Rabbit Ab, supplied by Biotium, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti rabbit antibodies
    Correlation of activities of <t>caspase</t> 9 and 3 with their active forms and PARP ( A ) HepG2 cells were incubated with 2 mM MNPE for an initial 2 h, changed to fresh medium, and incubated for an additional 15 h. With etoposide treatment, 0.4 mM etoposide (Etop) was present all the time. MNPE was added to 2 mM at 15 h after etoposide and incubated further for 2 h. ( B ) Approx. 10 μg of protein was separated by SDS/PAGE and blotted on to a Hybond-P membrane. Immunoblot analyses were performed using anti-(caspase 9), anti-(caspase 3) or anti-PARP antibodies. Typical data from triplicate experiments are shown. Sizes of proteins are shown in kDa. *, non-specific band.
    Anti Rabbit Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 817 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti
    Dopamine D1R stimulation activates <t>Fyn</t> but not <t>Src</t> in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p
    Rabbit Anti, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher anti rabbit pe ab
    Dopamine D1R stimulation activates <t>Fyn</t> but not <t>Src</t> in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p
    Anti Rabbit Pe Ab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Jackson Immuno cy3 anti rabbit antibody
    Dopamine D1R stimulation activates <t>Fyn</t> but not <t>Src</t> in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p
    Cy3 Anti Rabbit Antibody, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 90/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti flag antibody
    Dopamine D1R stimulation activates <t>Fyn</t> but not <t>Src</t> in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p
    Rabbit Anti Flag Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology rabbit anti gadph ab
    Dopamine D1R stimulation activates <t>Fyn</t> but not <t>Src</t> in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p
    Rabbit Anti Gadph Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies rabbit anti hc1q ab
    Binding properties of hIgG1 Fc-engineered variants toward C1q and complement factors. ELISA results showing binding of WT hIgG1 and Fc-engineered variants to ( A ) pure <t>hC1q,</t> ( B ) C1q present in human serum, and ( C ) pure C1q present in C1q-depleted human
    Rabbit Anti Hc1q Ab, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cocalico rabbit anti hcp1 ab
    T3SS and T6SS proteins are regulated by acid pH. (A) Immunoblot analyses of BopC, BopE, and GroEL in the pellet fractions of B. mallei SR1 grown in M9 broth with 4% glycerol adjusted to pH 5 or pH 7. GroEL served as a loading control. Data are representative of those from 2 independent experiments. (B) Representative confocal images of B. mallei Δ tssM ::GFP(pBHR2) grown for 3 h in LB broth (Lennox) adjusted to pH 5 or pH 7. B. mallei Δ tssM ::GFP(pBHR2-VirAG) grown in broth at pH 7 served as a positive control. All images were taken under the same laser power and gain. Data are representative of those from 2 independent experiments. Bar = 10 μm. (C) Immunoblot analysis of TssM and DnaK expression when B. mallei SR1 was grown in broth adjusted to pH 5 or pH 7 for 24 h. DnaK served as a loading control. Data are representative of those from 2 independent experiments. (D) Immunoblot analysis of <t>Hcp1</t> and GroEL expression when B. mallei SR1 was grown at pH 5 or pH 7 for 24 h. Hcp1 was not expressed when B. mallei DDA0746 (Δ virG ) or B. mallei DDA0742 (Δ hcp1 ) was grown at pH 5, whereas B. mallei SR1(pBHR2- virAG ) expressed Hcp1 when it was grown at pH 7. GroEL served as a loading control. Data are representative of those from 2 independent experiments. (E) Immunoblot analysis of Hcp1 and GroEL expression when B. pseudomallei AI was grown at pH 5 or pH 7 for 7 h. GroEL served as a loading control. Data are representative of those from 2 independent experiments.
    Rabbit Anti Hcp1 Ab, supplied by Cocalico, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    FUJIFILM rabbit anti iba1 antibodies
    Astrocyte-derived Shh exacerbate EAE lesion formation and disease severity. EAE was induced in both Glast-Cre ERT2 ; Shh Flox/Flox (Shh ACKO ) and Shh Flox/Flox (Shh ctrl ) mice (n=6 mice in each group). ( A-H ) Mice were sacrificed 32 days later. ( A ) Spinal cord cross sections were immunostained with anti-CD45 antibodies to identify leucocytes. Representative confocal images are shown. ( B ) Leucocyte infiltration was quantified as the number of CD45+cells/mm². ( C ) Spinal cord cross sections were immunostained with <t>anti-Iba1</t> antibodies to identify microglia. ( D ) Microglia activation was quantified as the Iba1+ surface area. ( E ) Activated Astrocytes were visualized after GFAP staining of Spinal cord cross sections. ( F ) Astrocyte activation was quantified as the Gfap+ surface area. ( G ) Spinal cord cross sections were immunostained with anti-Mbp antibodies to identify myelin. ( H ) Myelination was quantified as the MBP+ surface area. ( I ) Mice were scored daily from day 7 until the end of the experiment at day 32 after sensitization on a standard 5-point scale, nonlinear regression (Boltzmann sigmoidal). *: p≤0.05; **: p≤0.01; NS: not significant; Mann Withney test.
    Rabbit Anti Iba1 Antibodies, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 85/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology rabbit anti irf4 ab
    <t>IRF4</t> binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was
    Rabbit Anti Irf4 Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology rabbit polyclonal ab
    <t>IRF4</t> binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was
    Rabbit Polyclonal Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology rabbit anti c3 ab
    <t>IRF4</t> binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was
    Rabbit Anti C3 Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Trinity Biotech rabbit anti camp ab
    <t>IRF4</t> binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was
    Rabbit Anti Camp Ab, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc rabbit anti h3k9me2 ab
    <t>IRF4</t> binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was
    Rabbit Anti H3k9me2 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam rabbit anti kim1 ab
    <t>IRF4</t> binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was
    Rabbit Anti Kim1 Ab, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson rabbit anti s100b ab
    Effects of A7R KO on BBB disorders induced by gp41-I90 and METH. cBMECs were isolated from WT and A7R KO mice treated with gp41-I90 (GP41) or METH as described in our recent publications [ 9 , 49 ]. Cells without treatment were used as a control. Triple staining (TS) was done by DAPI (blue)/antibodies against CD146 (FITC/green) and <t>S100B</t> (for brain) (rhodamine/red). a : CECs (CD146 + /DAPI + ), and b : cBMECs (CD146 + /S100B + /DAPI + ). Cells were counted with six random fields. (* P
    Rabbit Anti S100b Ab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti cav1 rabbit ab
    Impaired adhesion to ICAM-1 under conditions of fluid shear stress in the absence of <t>Cav1.</t> ( A ) Static adhesion of WT (filled bars) versus Cav1-KO (open bars) CD8 T cells to ICAM-1. CD8 T cells were stimulated with 50 nM PdBu, 1 μg/ml anti-CD3 Ab, or 5 μg/ml SDF-1α before the start of the assay. Fluid shear flow rates were set at 0.3 dyne/cm 2 . ( B ) Rolling rates of the cells were analyzed in parallel with ( C ) the number of adherent cells. Data are shown as mean ± SEM of data pooled from two independent experiments, each performed in triplicate on two mice per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test). ( D ) Cav1-WT or Cav1-KO OT-1 CD8 T cells were incubated for 30 min with chimeric ICAM-1 in HEPES buffer alone or supplemented with 100 nM Mg 2+ , or stimulated with PdBu, N4 peptide, anti-CD3, or anti-CD3 plus anti-CD28, as indicated. ICAM-1 bound to T cells was detected by staining with human IgG-FITC. ICAM-1 MFI ± SEM from one of three independent experiments. ( E ) Representative histograms from each condition, as indicated. ns, not significant.
    Anti Cav1 Rabbit Ab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc anti rabbit polyclonal ab
    Impaired adhesion to ICAM-1 under conditions of fluid shear stress in the absence of <t>Cav1.</t> ( A ) Static adhesion of WT (filled bars) versus Cav1-KO (open bars) CD8 T cells to ICAM-1. CD8 T cells were stimulated with 50 nM PdBu, 1 μg/ml anti-CD3 Ab, or 5 μg/ml SDF-1α before the start of the assay. Fluid shear flow rates were set at 0.3 dyne/cm 2 . ( B ) Rolling rates of the cells were analyzed in parallel with ( C ) the number of adherent cells. Data are shown as mean ± SEM of data pooled from two independent experiments, each performed in triplicate on two mice per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test). ( D ) Cav1-WT or Cav1-KO OT-1 CD8 T cells were incubated for 30 min with chimeric ICAM-1 in HEPES buffer alone or supplemented with 100 nM Mg 2+ , or stimulated with PdBu, N4 peptide, anti-CD3, or anti-CD3 plus anti-CD28, as indicated. ICAM-1 bound to T cells was detected by staining with human IgG-FITC. ICAM-1 MFI ± SEM from one of three independent experiments. ( E ) Representative histograms from each condition, as indicated. ns, not significant.
    Anti Rabbit Polyclonal Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Enzo Biochem anti rabbit secondary ab
    Impaired adhesion to ICAM-1 under conditions of fluid shear stress in the absence of <t>Cav1.</t> ( A ) Static adhesion of WT (filled bars) versus Cav1-KO (open bars) CD8 T cells to ICAM-1. CD8 T cells were stimulated with 50 nM PdBu, 1 μg/ml anti-CD3 Ab, or 5 μg/ml SDF-1α before the start of the assay. Fluid shear flow rates were set at 0.3 dyne/cm 2 . ( B ) Rolling rates of the cells were analyzed in parallel with ( C ) the number of adherent cells. Data are shown as mean ± SEM of data pooled from two independent experiments, each performed in triplicate on two mice per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test). ( D ) Cav1-WT or Cav1-KO OT-1 CD8 T cells were incubated for 30 min with chimeric ICAM-1 in HEPES buffer alone or supplemented with 100 nM Mg 2+ , or stimulated with PdBu, N4 peptide, anti-CD3, or anti-CD3 plus anti-CD28, as indicated. ICAM-1 bound to T cells was detected by staining with human IgG-FITC. ICAM-1 MFI ± SEM from one of three independent experiments. ( E ) Representative histograms from each condition, as indicated. ns, not significant.
    Anti Rabbit Secondary Ab, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology rabbit anti flag antibody
    Requirement of soluble fraction for Mpo1-dependent FA α-oxidation. (A and B) Total cell lysates (100 μg) were prepared from BY4741 (wild type; WT) and 4378 ( mpo1 Δ) cells harboring the pAKNF316 (vector) or pNK46 (3×FLAG-MPO1) plasmid and incubated with 8 nCi (375 nM) 2-[9,10- 3 H]OH C 16:0 -COOH at 37°C for 2 h. After the reaction, lipids were extracted. Lipids were separated by normal-phase TLC and detected by autoradiography (A). Lipids were subjected to transmethylation, and the resulting FA methyl esters were separated by reverse-phase TLC together with C 14:0 and C 16:0 FA methyl ester standards, and detected by autoradiography (B). (C) Total cell lysates (T) were prepared from BY4741 (WT) and 4378 ( mpo1 Δ) cells and separated into soluble (S) and membrane (M) fractions by ultracentrifugation. Total cell lysates (100 μg), soluble fractions (60 μg), and membrane fractions (40 μg) prepared from BY4741 and 4378 cells were incubated with 8 nCi 2-[9,10- 3 H]OH C 16:0 -COOH at 37°C for 2 h. After the reactions, lipids were extracted, separated by normal-phase TLC, and detected by autoradiography. (D) Total cell lysates were prepared from 4378 cells bearing the pNK46 (3×FLAG-MPO1) plasmid and separated into soluble (S) and membrane (M) fractions by ultracentrifugation, followed by immunoblotting with <t>anti-FLAG,</t> anti-Pgk1 (soluble protein marker), and <t>anti-Pma1</t> (membrane protein marker).
    Rabbit Anti Flag Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc rabbit anti myd88 antibody
    SAHA attenuated KA-induced mRNA expression of TLR4, <t>MYD88,</t> NF-κB and IL-1β in hippocampus. In hippocampus CA1 region, mRNA expression of TLR4, MYD88, NF-κB and IL-1β were measured with real-time Quantitative PCR at 2, 6 h, 1 d and 2 d after KA injection. Relative quantification data were expressed as mean ± SD (n = 3). # P
    Rabbit Anti Myd88 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Correlation of activities of caspase 9 and 3 with their active forms and PARP ( A ) HepG2 cells were incubated with 2 mM MNPE for an initial 2 h, changed to fresh medium, and incubated for an additional 15 h. With etoposide treatment, 0.4 mM etoposide (Etop) was present all the time. MNPE was added to 2 mM at 15 h after etoposide and incubated further for 2 h. ( B ) Approx. 10 μg of protein was separated by SDS/PAGE and blotted on to a Hybond-P membrane. Immunoblot analyses were performed using anti-(caspase 9), anti-(caspase 3) or anti-PARP antibodies. Typical data from triplicate experiments are shown. Sizes of proteins are shown in kDa. *, non-specific band.

    Journal: Biochemical Journal

    Article Title: An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation

    doi: 10.1042/BJ20042067

    Figure Lengend Snippet: Correlation of activities of caspase 9 and 3 with their active forms and PARP ( A ) HepG2 cells were incubated with 2 mM MNPE for an initial 2 h, changed to fresh medium, and incubated for an additional 15 h. With etoposide treatment, 0.4 mM etoposide (Etop) was present all the time. MNPE was added to 2 mM at 15 h after etoposide and incubated further for 2 h. ( B ) Approx. 10 μg of protein was separated by SDS/PAGE and blotted on to a Hybond-P membrane. Immunoblot analyses were performed using anti-(caspase 9), anti-(caspase 3) or anti-PARP antibodies. Typical data from triplicate experiments are shown. Sizes of proteins are shown in kDa. *, non-specific band.

    Article Snippet: The membranes were then incubated with a mouse anti-(cytochrome c ) Ig (1:1000 dilution) (Pharmingen, San Diego, CA, U.S.A.), rabbit anti-(caspase 3) IgG (H-277, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), rabbit anti-(human caspase 9) Ig (Cell Signaling Technology, Beverly, MA, U.S.A.), or rabbit anti-PARP IgG (H-250, Santa Cruz Biotechnology) for 16 h at 4 °C.

    Techniques: Incubation, SDS Page

    Comparison of activities of caspase 9 and 3 between etoposide- and singlet-oxygen-induced cell death HepG2 cells were incubated with 2 mM MNPE for an initial 2 h, changed to fresh medium, and incubated for an additional 15 h. For etoposide treatment, 0.5 mM etoposide (Etop) was present at all times. A cellular lysate was prepared at the indicated time points. Activities of caspase 9 ( A ) and 3 ( B ) were assayed using specific substrates.

    Journal: Biochemical Journal

    Article Title: An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation

    doi: 10.1042/BJ20042067

    Figure Lengend Snippet: Comparison of activities of caspase 9 and 3 between etoposide- and singlet-oxygen-induced cell death HepG2 cells were incubated with 2 mM MNPE for an initial 2 h, changed to fresh medium, and incubated for an additional 15 h. For etoposide treatment, 0.5 mM etoposide (Etop) was present at all times. A cellular lysate was prepared at the indicated time points. Activities of caspase 9 ( A ) and 3 ( B ) were assayed using specific substrates.

    Article Snippet: The membranes were then incubated with a mouse anti-(cytochrome c ) Ig (1:1000 dilution) (Pharmingen, San Diego, CA, U.S.A.), rabbit anti-(caspase 3) IgG (H-277, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), rabbit anti-(human caspase 9) Ig (Cell Signaling Technology, Beverly, MA, U.S.A.), or rabbit anti-PARP IgG (H-250, Santa Cruz Biotechnology) for 16 h at 4 °C.

    Techniques: Incubation

    Effects of singlet oxygen and NO on caspase activities from cells pre-treated with etoposide, and the effects of reduction with DTT ( A )–( C ) Apoptosis was induced by 0.4 mM etoposide in HepG2 cells. Activities of caspase 9 and 3 were measured in cytosolic fractions of the cells that had been treated as follows. ( A ) Cytosolic fractions were incubated further with 2 mM MNPE or 2 mM SNAP for 2 h at 37 °C. ( B ) Cytosolic fractions were treated with various concentrations of MNPE for 2 h at 37 °C. ( C ) After treatment of the cytosolic fraction with either 2 mM MNPE or 2 mM SNAP as in ( A ), cytosolic fractions were incubated further with 10 mM DTT for 1 h at 37 °C. ( D ) The cytosolic fraction was prepared from untreated HepG2 cells. The caspase cascade was activated by adding activators, cytochrome c , dATP and MgCl 2 . Effects of 2 mM MNPE or 2 mM SNAP on caspase activities were examined in samples by co-incubation with these activators.

    Journal: Biochemical Journal

    Article Title: An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation

    doi: 10.1042/BJ20042067

    Figure Lengend Snippet: Effects of singlet oxygen and NO on caspase activities from cells pre-treated with etoposide, and the effects of reduction with DTT ( A )–( C ) Apoptosis was induced by 0.4 mM etoposide in HepG2 cells. Activities of caspase 9 and 3 were measured in cytosolic fractions of the cells that had been treated as follows. ( A ) Cytosolic fractions were incubated further with 2 mM MNPE or 2 mM SNAP for 2 h at 37 °C. ( B ) Cytosolic fractions were treated with various concentrations of MNPE for 2 h at 37 °C. ( C ) After treatment of the cytosolic fraction with either 2 mM MNPE or 2 mM SNAP as in ( A ), cytosolic fractions were incubated further with 10 mM DTT for 1 h at 37 °C. ( D ) The cytosolic fraction was prepared from untreated HepG2 cells. The caspase cascade was activated by adding activators, cytochrome c , dATP and MgCl 2 . Effects of 2 mM MNPE or 2 mM SNAP on caspase activities were examined in samples by co-incubation with these activators.

    Article Snippet: The membranes were then incubated with a mouse anti-(cytochrome c ) Ig (1:1000 dilution) (Pharmingen, San Diego, CA, U.S.A.), rabbit anti-(caspase 3) IgG (H-277, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), rabbit anti-(human caspase 9) Ig (Cell Signaling Technology, Beverly, MA, U.S.A.), or rabbit anti-PARP IgG (H-250, Santa Cruz Biotechnology) for 16 h at 4 °C.

    Techniques: Incubation

    Effects of pre-treatment of cells with MNPE on caspase activation in the cell-free system HepG2 cells were pre-treated without (−) or with (+) 2 mM MNPE for 2 h, and cytosolic fractions were prepared immediately. Caspases in the fractions were activated by adding activators, cytochrome c , dATP and MgCl 2 . ( A ) Activities of caspase 9 and 3 were measured in triplicate. ( B ) Approx. 10 μg of protein was separated by SDS/PAGE and blotted on to the Hybond-P membrane. Immunoblot analyses were performed using the anti-(caspase 9), anti-(caspase 3) or anti-(cytochrome c ) antibody. Sizes of proteins are shown in kDa. *, non-specific band. Typical data of triplicate experiments are shown.

    Journal: Biochemical Journal

    Article Title: An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation

    doi: 10.1042/BJ20042067

    Figure Lengend Snippet: Effects of pre-treatment of cells with MNPE on caspase activation in the cell-free system HepG2 cells were pre-treated without (−) or with (+) 2 mM MNPE for 2 h, and cytosolic fractions were prepared immediately. Caspases in the fractions were activated by adding activators, cytochrome c , dATP and MgCl 2 . ( A ) Activities of caspase 9 and 3 were measured in triplicate. ( B ) Approx. 10 μg of protein was separated by SDS/PAGE and blotted on to the Hybond-P membrane. Immunoblot analyses were performed using the anti-(caspase 9), anti-(caspase 3) or anti-(cytochrome c ) antibody. Sizes of proteins are shown in kDa. *, non-specific band. Typical data of triplicate experiments are shown.

    Article Snippet: The membranes were then incubated with a mouse anti-(cytochrome c ) Ig (1:1000 dilution) (Pharmingen, San Diego, CA, U.S.A.), rabbit anti-(caspase 3) IgG (H-277, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), rabbit anti-(human caspase 9) Ig (Cell Signaling Technology, Beverly, MA, U.S.A.), or rabbit anti-PARP IgG (H-250, Santa Cruz Biotechnology) for 16 h at 4 °C.

    Techniques: Activation Assay, SDS Page

    Dopamine D1R stimulation activates Fyn but not Src in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    doi: 10.3389/fnmol.2017.00273

    Figure Lengend Snippet: Dopamine D1R stimulation activates Fyn but not Src in the DMS but not in the DLS or NAc. (A) Schematic representation of DMS, DLS, and NAc dissection. (B–D) SKF81297 (SKF, 5 mg/kg) or vehicle (2% DMSO) was systemically administered (i.p.). The DMS (B) , DLS (C) , and NAc (D) were collected 15 min after drug administration. The level of Tyr420 phosphorylation (pFyn) (upper panel), Fyn (middle panel), and GAPDH, which was used as a loading control (bottom panel) were determined by western blot analysis. Data are presented as the densitometry value of the pFyn divided by the densitometry values of Fyn ± SEM and expressed as % of vehicle. (E,F) Mouse striatal sections were treated with SKF81297 (SKF, 10 μM) or vehicle (0.1% DMSO) for 15 min. The DMS was dissected and Fyn or Src were immunoprecipitated using anti-Fyn (IP Fyn) (E) or anti-Src (IP Src) (F) antibodies. Mouse IgG was used as a negative control (IP IgG). Fyn and Src immunoreactivity (lower panels) as well as Tyr417/420[Src/Fyn] phosphorylation (upper panels) were determined by westernblot analysis. Protein levels in the total lysates were measured in parallel (Input). Two-tailed t -test. (B–D) *** p

    Article Snippet: Rabbit anti-[pY1472]GluN2B antibodies and rabbit anti-[pY418/420]Src/Fyn antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Dissection, Western Blot, Immunoprecipitation, Negative Control, Two Tailed Test

    Binding properties of hIgG1 Fc-engineered variants toward C1q and complement factors. ELISA results showing binding of WT hIgG1 and Fc-engineered variants to ( A ) pure hC1q, ( B ) C1q present in human serum, and ( C ) pure C1q present in C1q-depleted human

    Journal: The Journal of Immunology Author Choice

    Article Title: Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

    doi: 10.4049/jimmunol.1401218

    Figure Lengend Snippet: Binding properties of hIgG1 Fc-engineered variants toward C1q and complement factors. ELISA results showing binding of WT hIgG1 and Fc-engineered variants to ( A ) pure hC1q, ( B ) C1q present in human serum, and ( C ) pure C1q present in C1q-depleted human

    Article Snippet: Bound hC1q was detected using a rabbit anti-hC1q Ab (Dako) followed by biotinylated sheep anti-rabbit IgG (locally produced) and subsequently alkaline phosphatase–conjugated streptavidin (GE Healthcare).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

    T3SS and T6SS proteins are regulated by acid pH. (A) Immunoblot analyses of BopC, BopE, and GroEL in the pellet fractions of B. mallei SR1 grown in M9 broth with 4% glycerol adjusted to pH 5 or pH 7. GroEL served as a loading control. Data are representative of those from 2 independent experiments. (B) Representative confocal images of B. mallei Δ tssM ::GFP(pBHR2) grown for 3 h in LB broth (Lennox) adjusted to pH 5 or pH 7. B. mallei Δ tssM ::GFP(pBHR2-VirAG) grown in broth at pH 7 served as a positive control. All images were taken under the same laser power and gain. Data are representative of those from 2 independent experiments. Bar = 10 μm. (C) Immunoblot analysis of TssM and DnaK expression when B. mallei SR1 was grown in broth adjusted to pH 5 or pH 7 for 24 h. DnaK served as a loading control. Data are representative of those from 2 independent experiments. (D) Immunoblot analysis of Hcp1 and GroEL expression when B. mallei SR1 was grown at pH 5 or pH 7 for 24 h. Hcp1 was not expressed when B. mallei DDA0746 (Δ virG ) or B. mallei DDA0742 (Δ hcp1 ) was grown at pH 5, whereas B. mallei SR1(pBHR2- virAG ) expressed Hcp1 when it was grown at pH 7. GroEL served as a loading control. Data are representative of those from 2 independent experiments. (E) Immunoblot analysis of Hcp1 and GroEL expression when B. pseudomallei AI was grown at pH 5 or pH 7 for 7 h. GroEL served as a loading control. Data are representative of those from 2 independent experiments.

    Journal: Infection and Immunity

    Article Title: pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells

    doi: 10.1128/IAI.00586-16

    Figure Lengend Snippet: T3SS and T6SS proteins are regulated by acid pH. (A) Immunoblot analyses of BopC, BopE, and GroEL in the pellet fractions of B. mallei SR1 grown in M9 broth with 4% glycerol adjusted to pH 5 or pH 7. GroEL served as a loading control. Data are representative of those from 2 independent experiments. (B) Representative confocal images of B. mallei Δ tssM ::GFP(pBHR2) grown for 3 h in LB broth (Lennox) adjusted to pH 5 or pH 7. B. mallei Δ tssM ::GFP(pBHR2-VirAG) grown in broth at pH 7 served as a positive control. All images were taken under the same laser power and gain. Data are representative of those from 2 independent experiments. Bar = 10 μm. (C) Immunoblot analysis of TssM and DnaK expression when B. mallei SR1 was grown in broth adjusted to pH 5 or pH 7 for 24 h. DnaK served as a loading control. Data are representative of those from 2 independent experiments. (D) Immunoblot analysis of Hcp1 and GroEL expression when B. mallei SR1 was grown at pH 5 or pH 7 for 24 h. Hcp1 was not expressed when B. mallei DDA0746 (Δ virG ) or B. mallei DDA0742 (Δ hcp1 ) was grown at pH 5, whereas B. mallei SR1(pBHR2- virAG ) expressed Hcp1 when it was grown at pH 7. GroEL served as a loading control. Data are representative of those from 2 independent experiments. (E) Immunoblot analysis of Hcp1 and GroEL expression when B. pseudomallei AI was grown at pH 5 or pH 7 for 7 h. GroEL served as a loading control. Data are representative of those from 2 independent experiments.

    Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% nonfat milk for 2 h. Incubation with rabbit anti-BopC Ab (kindly given by Sunee Korbsrisate, Mahidol University, Bangkok, Thailand), rat anti-BopE Ab (Cocalico), mouse anti-TssM Ab , rabbit anti-Hcp1 Ab (Cocalico), mouse anti-DnaK Ab (Stressgen), or rabbit anti-GroEL Ab (Stressgen) occurred at 4°C overnight.

    Techniques: Positive Control, Expressing

    Astrocyte-derived Shh exacerbate EAE lesion formation and disease severity. EAE was induced in both Glast-Cre ERT2 ; Shh Flox/Flox (Shh ACKO ) and Shh Flox/Flox (Shh ctrl ) mice (n=6 mice in each group). ( A-H ) Mice were sacrificed 32 days later. ( A ) Spinal cord cross sections were immunostained with anti-CD45 antibodies to identify leucocytes. Representative confocal images are shown. ( B ) Leucocyte infiltration was quantified as the number of CD45+cells/mm². ( C ) Spinal cord cross sections were immunostained with anti-Iba1 antibodies to identify microglia. ( D ) Microglia activation was quantified as the Iba1+ surface area. ( E ) Activated Astrocytes were visualized after GFAP staining of Spinal cord cross sections. ( F ) Astrocyte activation was quantified as the Gfap+ surface area. ( G ) Spinal cord cross sections were immunostained with anti-Mbp antibodies to identify myelin. ( H ) Myelination was quantified as the MBP+ surface area. ( I ) Mice were scored daily from day 7 until the end of the experiment at day 32 after sensitization on a standard 5-point scale, nonlinear regression (Boltzmann sigmoidal). *: p≤0.05; **: p≤0.01; NS: not significant; Mann Withney test.

    Journal: bioRxiv

    Article Title: Endothelial cell response to Hedgehog ligands depends on their processing

    doi: 10.1101/2020.03.03.974444

    Figure Lengend Snippet: Astrocyte-derived Shh exacerbate EAE lesion formation and disease severity. EAE was induced in both Glast-Cre ERT2 ; Shh Flox/Flox (Shh ACKO ) and Shh Flox/Flox (Shh ctrl ) mice (n=6 mice in each group). ( A-H ) Mice were sacrificed 32 days later. ( A ) Spinal cord cross sections were immunostained with anti-CD45 antibodies to identify leucocytes. Representative confocal images are shown. ( B ) Leucocyte infiltration was quantified as the number of CD45+cells/mm². ( C ) Spinal cord cross sections were immunostained with anti-Iba1 antibodies to identify microglia. ( D ) Microglia activation was quantified as the Iba1+ surface area. ( E ) Activated Astrocytes were visualized after GFAP staining of Spinal cord cross sections. ( F ) Astrocyte activation was quantified as the Gfap+ surface area. ( G ) Spinal cord cross sections were immunostained with anti-Mbp antibodies to identify myelin. ( H ) Myelination was quantified as the MBP+ surface area. ( I ) Mice were scored daily from day 7 until the end of the experiment at day 32 after sensitization on a standard 5-point scale, nonlinear regression (Boltzmann sigmoidal). *: p≤0.05; **: p≤0.01; NS: not significant; Mann Withney test.

    Article Snippet: Iba1+ microglia was identified using rabbit anti-Iba1 antibodies (FUJUFILM Wako chemicals, cat#019-19741).

    Techniques: Derivative Assay, Mouse Assay, Activation Assay, Staining

    IRF4 binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Fas Apoptosis Inhibitory Molecule Expression in B Cells Is Regulated through IRF4 in a Feed-Forward Mechanism

    doi: 10.4049/jimmunol.0901988

    Figure Lengend Snippet: IRF4 binds the Faim promoter in stimulated primary B cells. Primary B cells were stimulated by CD40L for 48 h alone (0 h) or were stimulated by CD40L for 48 h during which time anti-Ig was added for the final number of hours indicated and ChIP assay was

    Article Snippet: Rabbit anti-IRF4 Ab was obtained from Santa Cruz Biotechnology.

    Techniques: Chromatin Immunoprecipitation

    Stimulated FAIM expression is preceded by induction of IRF4 expression in primary B cells. A , Primary B cells were stimulated by CD40L for 48 h alone (NS), or were stimulated by CD40L for 48 h during which time either anti-Ig, LPS, or IL-4 was added for

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Fas Apoptosis Inhibitory Molecule Expression in B Cells Is Regulated through IRF4 in a Feed-Forward Mechanism

    doi: 10.4049/jimmunol.0901988

    Figure Lengend Snippet: Stimulated FAIM expression is preceded by induction of IRF4 expression in primary B cells. A , Primary B cells were stimulated by CD40L for 48 h alone (NS), or were stimulated by CD40L for 48 h during which time either anti-Ig, LPS, or IL-4 was added for

    Article Snippet: Rabbit anti-IRF4 Ab was obtained from Santa Cruz Biotechnology.

    Techniques: Expressing

    IRF4 plays a nonredundant role in up-regulating FAIM expression. Follicular B cells from IRF4-null mice and from littermate control mice were sort-purified as described in Materials and Methods and then harvested before stimulation, or were stimulated

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Fas Apoptosis Inhibitory Molecule Expression in B Cells Is Regulated through IRF4 in a Feed-Forward Mechanism

    doi: 10.4049/jimmunol.0901988

    Figure Lengend Snippet: IRF4 plays a nonredundant role in up-regulating FAIM expression. Follicular B cells from IRF4-null mice and from littermate control mice were sort-purified as described in Materials and Methods and then harvested before stimulation, or were stimulated

    Article Snippet: Rabbit anti-IRF4 Ab was obtained from Santa Cruz Biotechnology.

    Techniques: Expressing, Mouse Assay, Purification

    Effects of A7R KO on BBB disorders induced by gp41-I90 and METH. cBMECs were isolated from WT and A7R KO mice treated with gp41-I90 (GP41) or METH as described in our recent publications [ 9 , 49 ]. Cells without treatment were used as a control. Triple staining (TS) was done by DAPI (blue)/antibodies against CD146 (FITC/green) and S100B (for brain) (rhodamine/red). a : CECs (CD146 + /DAPI + ), and b : cBMECs (CD146 + /S100B + /DAPI + ). Cells were counted with six random fields. (* P

    Journal: BMC Infectious Diseases

    Article Title: Alpha7 nicotinic acetylcholine receptor is required for blood-brain barrier injury-related CNS disorders caused by Cryptococcus neoformans and HIV-1 associated comorbidity factors

    doi: 10.1186/s12879-015-1075-9

    Figure Lengend Snippet: Effects of A7R KO on BBB disorders induced by gp41-I90 and METH. cBMECs were isolated from WT and A7R KO mice treated with gp41-I90 (GP41) or METH as described in our recent publications [ 9 , 49 ]. Cells without treatment were used as a control. Triple staining (TS) was done by DAPI (blue)/antibodies against CD146 (FITC/green) and S100B (for brain) (rhodamine/red). a : CECs (CD146 + /DAPI + ), and b : cBMECs (CD146 + /S100B + /DAPI + ). Cells were counted with six random fields. (* P

    Article Snippet: A Fluoro-Jade B (FJB) staining kit was obtained from Histo-Chemo Inc. All primary antibodies (Ab) were purchased from the commercial sources: a rabbit anti-α7 nAChR Ab from Genescript (Piscataway, NJ); Anti-NF-κB/p65 mAb from Cell Signal Technology (Cell Signal Technology, #3033); an antibody against dimethyl-histone H3 (Lys9) from Millipore (Millipore, cat #.07–212), an anti-mouse CD146 Ab FITC-conjugated and a mouse anti-neuron (NeuN) Ab from eBiosciences (San Diego, CA); a mouse anti-CD44 Ab (sc-7297); a rabbit anti-CD54 Ab (ICAM-1, 250593) from Abbiotec (San Diego, CA); a rabbit anti-β-actin (sc-7210) and an anti-GFAP Ab from Santa Cruz Biotechnology (Santa Cruz, CA); an anti-mouse CD146 Ab FITC-conjugated from Biolegend (San Diego, CA), and a rabbit anti-S100B Ab from BD Biosciences.

    Techniques: Isolation, Mouse Assay, Staining

    Impaired adhesion to ICAM-1 under conditions of fluid shear stress in the absence of Cav1. ( A ) Static adhesion of WT (filled bars) versus Cav1-KO (open bars) CD8 T cells to ICAM-1. CD8 T cells were stimulated with 50 nM PdBu, 1 μg/ml anti-CD3 Ab, or 5 μg/ml SDF-1α before the start of the assay. Fluid shear flow rates were set at 0.3 dyne/cm 2 . ( B ) Rolling rates of the cells were analyzed in parallel with ( C ) the number of adherent cells. Data are shown as mean ± SEM of data pooled from two independent experiments, each performed in triplicate on two mice per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test). ( D ) Cav1-WT or Cav1-KO OT-1 CD8 T cells were incubated for 30 min with chimeric ICAM-1 in HEPES buffer alone or supplemented with 100 nM Mg 2+ , or stimulated with PdBu, N4 peptide, anti-CD3, or anti-CD3 plus anti-CD28, as indicated. ICAM-1 bound to T cells was detected by staining with human IgG-FITC. ICAM-1 MFI ± SEM from one of three independent experiments. ( E ) Representative histograms from each condition, as indicated. ns, not significant.

    Journal: The Journal of Immunology Author Choice

    Article Title: Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

    doi: 10.4049/jimmunol.1700431

    Figure Lengend Snippet: Impaired adhesion to ICAM-1 under conditions of fluid shear stress in the absence of Cav1. ( A ) Static adhesion of WT (filled bars) versus Cav1-KO (open bars) CD8 T cells to ICAM-1. CD8 T cells were stimulated with 50 nM PdBu, 1 μg/ml anti-CD3 Ab, or 5 μg/ml SDF-1α before the start of the assay. Fluid shear flow rates were set at 0.3 dyne/cm 2 . ( B ) Rolling rates of the cells were analyzed in parallel with ( C ) the number of adherent cells. Data are shown as mean ± SEM of data pooled from two independent experiments, each performed in triplicate on two mice per group. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test). ( D ) Cav1-WT or Cav1-KO OT-1 CD8 T cells were incubated for 30 min with chimeric ICAM-1 in HEPES buffer alone or supplemented with 100 nM Mg 2+ , or stimulated with PdBu, N4 peptide, anti-CD3, or anti-CD3 plus anti-CD28, as indicated. ICAM-1 bound to T cells was detected by staining with human IgG-FITC. ICAM-1 MFI ± SEM from one of three independent experiments. ( E ) Representative histograms from each condition, as indicated. ns, not significant.

    Article Snippet: Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences).

    Techniques: Flow Cytometry, Mouse Assay, Incubation, Staining

    Cav1 regulates CD8 T cell responses to Ag. ( A ) The frequency of CD69 + cells among Cav1-WT and Cav1-KO OT-1 CD8 T cells stimulated for 3 h with a titration of N4, T4, or G4 peptides. ( B and C ) Cav1-WT and Cav1-KO OT-1 CD8 T cells were stimulated and supernatants were assayed at 24 h for total IL-2 (B) and TNF-α (C) by ELISA. ( D ) Proliferation at 72 h of CTV-labeled OT-1 T cells is represented by the division index calculated using FlowJo software. Data are representative of two independent experiments, each performed on three mice per group. Error bars represent SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test, corrected for multiple comparisons using the Holm–Sidak method).

    Journal: The Journal of Immunology Author Choice

    Article Title: Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

    doi: 10.4049/jimmunol.1700431

    Figure Lengend Snippet: Cav1 regulates CD8 T cell responses to Ag. ( A ) The frequency of CD69 + cells among Cav1-WT and Cav1-KO OT-1 CD8 T cells stimulated for 3 h with a titration of N4, T4, or G4 peptides. ( B and C ) Cav1-WT and Cav1-KO OT-1 CD8 T cells were stimulated and supernatants were assayed at 24 h for total IL-2 (B) and TNF-α (C) by ELISA. ( D ) Proliferation at 72 h of CTV-labeled OT-1 T cells is represented by the division index calculated using FlowJo software. Data are representative of two independent experiments, each performed on three mice per group. Error bars represent SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student t test, corrected for multiple comparisons using the Holm–Sidak method).

    Article Snippet: Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences).

    Techniques: Titration, Enzyme-linked Immunosorbent Assay, Labeling, Software, Mouse Assay

    Impaired recruitment of F-actin and CD11a to the IS in the absence of Cav1. ImageStream analysis assessing conjugate formation of Cav1-WT or Cav1-KO OT-1 CD8 T cells with N4-pulsed APCs for the indicated times (0–20 min) is shown. Original magnification ×60. Conjugates were fixed, permeabilized, and stained for BODIPY-Fl (F-actin, green) and CD11a (yellow). ( A ) The four rows show representative cells at 20 min, with distribution of BODIPY-Fl and CD11a within the synapse formed between T cells (purple) and APCs (red) shown in merge columns 4 and 5, respectively. Graphs of the internalization method, previously described in ( 35 ), for Cav1-WT and Cav1-KO conjugates stained with ( B ) BODIPY-Fl and ( C ) CD11a are representative of three independent experiments.

    Journal: The Journal of Immunology Author Choice

    Article Title: Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

    doi: 10.4049/jimmunol.1700431

    Figure Lengend Snippet: Impaired recruitment of F-actin and CD11a to the IS in the absence of Cav1. ImageStream analysis assessing conjugate formation of Cav1-WT or Cav1-KO OT-1 CD8 T cells with N4-pulsed APCs for the indicated times (0–20 min) is shown. Original magnification ×60. Conjugates were fixed, permeabilized, and stained for BODIPY-Fl (F-actin, green) and CD11a (yellow). ( A ) The four rows show representative cells at 20 min, with distribution of BODIPY-Fl and CD11a within the synapse formed between T cells (purple) and APCs (red) shown in merge columns 4 and 5, respectively. Graphs of the internalization method, previously described in ( 35 ), for Cav1-WT and Cav1-KO conjugates stained with ( B ) BODIPY-Fl and ( C ) CD11a are representative of three independent experiments.

    Article Snippet: Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences).

    Techniques: Staining

    Impaired LFA-1–mediated CD8 T cell homotypic aggregation in the absence of Cav1. ( A ) Purified Cav1-WT (filled bars) and Cav1-KO (open bars) OT-1 CD8 T cells were activated with 50 ng/ml PdBu or 1 μg/ml anti-CD3 for 60 min and assessed for cell–cell aggregation. Data are shown as a mean ± SEM of data representing one of five independent experiments with each count performed in triplicate on two to four mice per group. ( B ) Expression levels of the indicated surface proteins in WT (gray shaded) and Cav1-KO (black line) naive OT-1 CD8 T cells. Data are representative of 25 mice per group. Naive CD8 T cells were cultured for 24 h and then ( C ) imaged and ( D ) assessed for cluster size using Volocity software. Data are pooled from two independent experiments with a minimum of 100 cells per condition (mean and SD). Scale bars, 5 μm. ( E ) Anti–LFA-1 blocking mAb was added at various time points or ( F ) at various concentrations, following 1 μM N4 peptide stimulation of Cav1-WT and Cav1-KO CD8 T cells. Inhibition of CD69 upregulation (percentage inhibition) was measured at 3 h. Data are representative of one of three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p = 0.0001 (Student t test); ns, not significant.

    Journal: The Journal of Immunology Author Choice

    Article Title: Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

    doi: 10.4049/jimmunol.1700431

    Figure Lengend Snippet: Impaired LFA-1–mediated CD8 T cell homotypic aggregation in the absence of Cav1. ( A ) Purified Cav1-WT (filled bars) and Cav1-KO (open bars) OT-1 CD8 T cells were activated with 50 ng/ml PdBu or 1 μg/ml anti-CD3 for 60 min and assessed for cell–cell aggregation. Data are shown as a mean ± SEM of data representing one of five independent experiments with each count performed in triplicate on two to four mice per group. ( B ) Expression levels of the indicated surface proteins in WT (gray shaded) and Cav1-KO (black line) naive OT-1 CD8 T cells. Data are representative of 25 mice per group. Naive CD8 T cells were cultured for 24 h and then ( C ) imaged and ( D ) assessed for cluster size using Volocity software. Data are pooled from two independent experiments with a minimum of 100 cells per condition (mean and SD). Scale bars, 5 μm. ( E ) Anti–LFA-1 blocking mAb was added at various time points or ( F ) at various concentrations, following 1 μM N4 peptide stimulation of Cav1-WT and Cav1-KO CD8 T cells. Inhibition of CD69 upregulation (percentage inhibition) was measured at 3 h. Data are representative of one of three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p = 0.0001 (Student t test); ns, not significant.

    Article Snippet: Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences).

    Techniques: Purification, Mouse Assay, Expressing, Cell Culture, Software, Blocking Assay, Inhibition

    Absence of Cav1 alters CD8 T cell morphology and polarization upon adhesion to ICAM-1. Cav1-WT and Cav1-KO OT-1 CD8 T cells were allowed to adhere for 30 min to glass slides with and without 3 μg/ml ICAM-1 coating. ( A ) Cells were stained with BODIPY-Fl (green) to define the cell perimeter. Cav1-WT and Cav1-KO CD8 T cells spreading on ICAM-1 were quantified in terms of ( B ) area, ( C ) longest axis, and ( D ) shape factor. For shape factor, a value of 1 is circular. (B–D) Small horizontal lines indicate the mean. ( E and F ) Cells were stained with Ezrin (green) and Lck (red). Merge images were sectioned (white line) from the leading edge, indicated by the asterisk, to the uropod, generating an RGB histogram using ImageJ software. The histogram line indicates the distribution and MFI of proteins throughout the cross-section of the cell. ( G ) A Pearson correlation coefficient was calculated by Volocity software, and p values were calculated by the Student t test. Data are representative of one of three independent experiments with a minimum of 100 cells per condition. Scale bars, 1 μm. **** p ≤ 0.001.

    Journal: The Journal of Immunology Author Choice

    Article Title: Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

    doi: 10.4049/jimmunol.1700431

    Figure Lengend Snippet: Absence of Cav1 alters CD8 T cell morphology and polarization upon adhesion to ICAM-1. Cav1-WT and Cav1-KO OT-1 CD8 T cells were allowed to adhere for 30 min to glass slides with and without 3 μg/ml ICAM-1 coating. ( A ) Cells were stained with BODIPY-Fl (green) to define the cell perimeter. Cav1-WT and Cav1-KO CD8 T cells spreading on ICAM-1 were quantified in terms of ( B ) area, ( C ) longest axis, and ( D ) shape factor. For shape factor, a value of 1 is circular. (B–D) Small horizontal lines indicate the mean. ( E and F ) Cells were stained with Ezrin (green) and Lck (red). Merge images were sectioned (white line) from the leading edge, indicated by the asterisk, to the uropod, generating an RGB histogram using ImageJ software. The histogram line indicates the distribution and MFI of proteins throughout the cross-section of the cell. ( G ) A Pearson correlation coefficient was calculated by Volocity software, and p values were calculated by the Student t test. Data are representative of one of three independent experiments with a minimum of 100 cells per condition. Scale bars, 1 μm. **** p ≤ 0.001.

    Article Snippet: Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences).

    Techniques: Staining, Software

    Cav1 is localized within soluble and insoluble membrane fractions and regulates cholesterol and lipid composition. ( A ) Cav1 and Cav2 protein expression was compared between WT and Cav1-KO CD8 T cell lysates by Western blot with anti-Cav1 and Cav2 Ab. Cell lysates were immunoprecipitated with anti-Cav1 and Western blots were probed sequentially with anti-Cav1 and Cav2 Ab. ( B ) Cell lysates were separated into subcellular fractions (C, cytoplasmic; MS, membrane soluble; NS, nuclear soluble; CB, chromatin bound; LCC, lipid-cytoskeletal complexes) and analyzed by Western blot with anti-Cav1 Ab. ( C ) Total sphingomyelin content in WT versus Cav1-KO CD8 T cells. ( D ) Sphingomyelin species 14:0 and 16:0 are represented as a percentage of total sphingomyelin. ( E ) Free cholesterol from WT and Cav1-KO CD8 T cells is representative of one of three independent experiments from a total of 12 biological samples of each genotype. ( F ) Total ceramide and ( G ) ceramide monohexamide content. Lipidomics data are pooled from three biological repeats. Data are shown as mean + SD. * p ≤ 0.05, ** p ≤ 0.01 (Student t test). ns, not significant.

    Journal: The Journal of Immunology Author Choice

    Article Title: Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells

    doi: 10.4049/jimmunol.1700431

    Figure Lengend Snippet: Cav1 is localized within soluble and insoluble membrane fractions and regulates cholesterol and lipid composition. ( A ) Cav1 and Cav2 protein expression was compared between WT and Cav1-KO CD8 T cell lysates by Western blot with anti-Cav1 and Cav2 Ab. Cell lysates were immunoprecipitated with anti-Cav1 and Western blots were probed sequentially with anti-Cav1 and Cav2 Ab. ( B ) Cell lysates were separated into subcellular fractions (C, cytoplasmic; MS, membrane soluble; NS, nuclear soluble; CB, chromatin bound; LCC, lipid-cytoskeletal complexes) and analyzed by Western blot with anti-Cav1 Ab. ( C ) Total sphingomyelin content in WT versus Cav1-KO CD8 T cells. ( D ) Sphingomyelin species 14:0 and 16:0 are represented as a percentage of total sphingomyelin. ( E ) Free cholesterol from WT and Cav1-KO CD8 T cells is representative of one of three independent experiments from a total of 12 biological samples of each genotype. ( F ) Total ceramide and ( G ) ceramide monohexamide content. Lipidomics data are pooled from three biological repeats. Data are shown as mean + SD. * p ≤ 0.05, ** p ≤ 0.01 (Student t test). ns, not significant.

    Article Snippet: Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Mass Spectrometry

    Requirement of soluble fraction for Mpo1-dependent FA α-oxidation. (A and B) Total cell lysates (100 μg) were prepared from BY4741 (wild type; WT) and 4378 ( mpo1 Δ) cells harboring the pAKNF316 (vector) or pNK46 (3×FLAG-MPO1) plasmid and incubated with 8 nCi (375 nM) 2-[9,10- 3 H]OH C 16:0 -COOH at 37°C for 2 h. After the reaction, lipids were extracted. Lipids were separated by normal-phase TLC and detected by autoradiography (A). Lipids were subjected to transmethylation, and the resulting FA methyl esters were separated by reverse-phase TLC together with C 14:0 and C 16:0 FA methyl ester standards, and detected by autoradiography (B). (C) Total cell lysates (T) were prepared from BY4741 (WT) and 4378 ( mpo1 Δ) cells and separated into soluble (S) and membrane (M) fractions by ultracentrifugation. Total cell lysates (100 μg), soluble fractions (60 μg), and membrane fractions (40 μg) prepared from BY4741 and 4378 cells were incubated with 8 nCi 2-[9,10- 3 H]OH C 16:0 -COOH at 37°C for 2 h. After the reactions, lipids were extracted, separated by normal-phase TLC, and detected by autoradiography. (D) Total cell lysates were prepared from 4378 cells bearing the pNK46 (3×FLAG-MPO1) plasmid and separated into soluble (S) and membrane (M) fractions by ultracentrifugation, followed by immunoblotting with anti-FLAG, anti-Pgk1 (soluble protein marker), and anti-Pma1 (membrane protein marker).

    Journal: Molecular and Cellular Biology

    Article Title: Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe2+-Dependent Manner

    doi: 10.1128/MCB.00428-18

    Figure Lengend Snippet: Requirement of soluble fraction for Mpo1-dependent FA α-oxidation. (A and B) Total cell lysates (100 μg) were prepared from BY4741 (wild type; WT) and 4378 ( mpo1 Δ) cells harboring the pAKNF316 (vector) or pNK46 (3×FLAG-MPO1) plasmid and incubated with 8 nCi (375 nM) 2-[9,10- 3 H]OH C 16:0 -COOH at 37°C for 2 h. After the reaction, lipids were extracted. Lipids were separated by normal-phase TLC and detected by autoradiography (A). Lipids were subjected to transmethylation, and the resulting FA methyl esters were separated by reverse-phase TLC together with C 14:0 and C 16:0 FA methyl ester standards, and detected by autoradiography (B). (C) Total cell lysates (T) were prepared from BY4741 (WT) and 4378 ( mpo1 Δ) cells and separated into soluble (S) and membrane (M) fractions by ultracentrifugation. Total cell lysates (100 μg), soluble fractions (60 μg), and membrane fractions (40 μg) prepared from BY4741 and 4378 cells were incubated with 8 nCi 2-[9,10- 3 H]OH C 16:0 -COOH at 37°C for 2 h. After the reactions, lipids were extracted, separated by normal-phase TLC, and detected by autoradiography. (D) Total cell lysates were prepared from 4378 cells bearing the pNK46 (3×FLAG-MPO1) plasmid and separated into soluble (S) and membrane (M) fractions by ultracentrifugation, followed by immunoblotting with anti-FLAG, anti-Pgk1 (soluble protein marker), and anti-Pma1 (membrane protein marker).

    Article Snippet: Rabbit anti-FLAG antibody (1:1,000 dilution) , goat anti-Pma1 antibody (1:2,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-Pgk1 polyclonal antibody (1:500 dilution) that was raised against a peptide (NH2 -CDANTKTVTDKEGIPAG-COOH) were used as the primary antibodies.

    Techniques: Plasmid Preparation, Incubation, Thin Layer Chromatography, Autoradiography, Marker

    SAHA attenuated KA-induced mRNA expression of TLR4, MYD88, NF-κB and IL-1β in hippocampus. In hippocampus CA1 region, mRNA expression of TLR4, MYD88, NF-κB and IL-1β were measured with real-time Quantitative PCR at 2, 6 h, 1 d and 2 d after KA injection. Relative quantification data were expressed as mean ± SD (n = 3). # P

    Journal: BMC Neuroscience

    Article Title: Histone deacetylase inhibitor SAHA attenuates post-seizure hippocampal microglia TLR4/MYD88 signaling and inhibits TLR4 gene expression via histone acetylation

    doi: 10.1186/s12868-016-0264-9

    Figure Lengend Snippet: SAHA attenuated KA-induced mRNA expression of TLR4, MYD88, NF-κB and IL-1β in hippocampus. In hippocampus CA1 region, mRNA expression of TLR4, MYD88, NF-κB and IL-1β were measured with real-time Quantitative PCR at 2, 6 h, 1 d and 2 d after KA injection. Relative quantification data were expressed as mean ± SD (n = 3). # P

    Article Snippet: The membranes were soaked in 5 % skimmed milk as blocking buffer for 2 h and then incubated with appropriate primary antibodies including mouse anti-TLR4 antibody (1:1000; Abcam), rabbit anti-MYD88 antibody (1:1000; Cell Signaling, Beverly, MA, USA), rabbit anti-NF-kB P65 antibody (1:2000; Proteintech), rabbit anti-IL-1β antibody (1:500, Proteintech) and mouse anti-β-actin (1:2000; Abcam), respectively, at 4 °C overnight.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection

    SAHA inhibited TLR4 signal pathway in KA-induced seizures. The protein expression of TLR4, MYD88, NF-κB P65 and IL-1β were examined by immunoblots at 2, 6 h, 1 d and 2 d after KA injection in hippocampus CA1 region. Specific bands were quantified by densitometry and data were expressed as mean ± SD (n = 3); # P

    Journal: BMC Neuroscience

    Article Title: Histone deacetylase inhibitor SAHA attenuates post-seizure hippocampal microglia TLR4/MYD88 signaling and inhibits TLR4 gene expression via histone acetylation

    doi: 10.1186/s12868-016-0264-9

    Figure Lengend Snippet: SAHA inhibited TLR4 signal pathway in KA-induced seizures. The protein expression of TLR4, MYD88, NF-κB P65 and IL-1β were examined by immunoblots at 2, 6 h, 1 d and 2 d after KA injection in hippocampus CA1 region. Specific bands were quantified by densitometry and data were expressed as mean ± SD (n = 3); # P

    Article Snippet: The membranes were soaked in 5 % skimmed milk as blocking buffer for 2 h and then incubated with appropriate primary antibodies including mouse anti-TLR4 antibody (1:1000; Abcam), rabbit anti-MYD88 antibody (1:1000; Cell Signaling, Beverly, MA, USA), rabbit anti-NF-kB P65 antibody (1:2000; Proteintech), rabbit anti-IL-1β antibody (1:500, Proteintech) and mouse anti-β-actin (1:2000; Abcam), respectively, at 4 °C overnight.

    Techniques: Expressing, Western Blot, Injection