rat anti c3  (Hycult Biotech)


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    Hycult Biotech rat anti c3
    Rat Anti C3, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
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    rat anti c3 - by Bioz Stars, 2024-04
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    rat anti c4 mab  (Hycult Biotech)


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    Hycult Biotech rat anti c4 mab
    <t>C4</t> protein localization after retinal detachment for sequencing validation. ( A ) QRT-PCR analysis of C4b mRNA levels in naïve and detached RPE at 1 dprd and 7 dprd. ( B ) Representative immunoblot of complement C4 in the soluble fluid fraction from C57BL6/J mouse eyes under naïve, contralateral (fellow at 7 dprd), and detached (7 dprd) conditions with protein samples separated under non-reducing conditions. Blots are representative of 3 independent animals in each condition. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Dunnett’s post hoc test. * p < 0.05; **** p < 0.0001. ( C ) IF analysis of ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. Cell nuclei staining (blue), and IF of anti-RPE65 mAb (green) and anti-C4 serum (red) are shown. ( D ) IF analysis of C4 protein in retinal and subretinal Iba-1 + immune cells in ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. IF of anti-C4 mAb (green), anti-Iba1 mAb (red, a microglia and monocyte/macrophage marker ). Staining cell nuclei (blue) is shown in the merged images (right column). Images were acquired using the Leica STELLARIS 8 FALCON Confocal Microscope, 40x objective
    Rat Anti C4 Mab, supplied by Hycult Biotech, 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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    rat anti c4 mab - by Bioz Stars, 2024-04
    86/100 stars

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    1) Product Images from "The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment"

    Article Title: The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-024-03062-2

    C4 protein localization after retinal detachment for sequencing validation. ( A ) QRT-PCR analysis of C4b mRNA levels in naïve and detached RPE at 1 dprd and 7 dprd. ( B ) Representative immunoblot of complement C4 in the soluble fluid fraction from C57BL6/J mouse eyes under naïve, contralateral (fellow at 7 dprd), and detached (7 dprd) conditions with protein samples separated under non-reducing conditions. Blots are representative of 3 independent animals in each condition. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Dunnett’s post hoc test. * p < 0.05; **** p < 0.0001. ( C ) IF analysis of ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. Cell nuclei staining (blue), and IF of anti-RPE65 mAb (green) and anti-C4 serum (red) are shown. ( D ) IF analysis of C4 protein in retinal and subretinal Iba-1 + immune cells in ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. IF of anti-C4 mAb (green), anti-Iba1 mAb (red, a microglia and monocyte/macrophage marker ). Staining cell nuclei (blue) is shown in the merged images (right column). Images were acquired using the Leica STELLARIS 8 FALCON Confocal Microscope, 40x objective
    Figure Legend Snippet: C4 protein localization after retinal detachment for sequencing validation. ( A ) QRT-PCR analysis of C4b mRNA levels in naïve and detached RPE at 1 dprd and 7 dprd. ( B ) Representative immunoblot of complement C4 in the soluble fluid fraction from C57BL6/J mouse eyes under naïve, contralateral (fellow at 7 dprd), and detached (7 dprd) conditions with protein samples separated under non-reducing conditions. Blots are representative of 3 independent animals in each condition. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Dunnett’s post hoc test. * p < 0.05; **** p < 0.0001. ( C ) IF analysis of ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. Cell nuclei staining (blue), and IF of anti-RPE65 mAb (green) and anti-C4 serum (red) are shown. ( D ) IF analysis of C4 protein in retinal and subretinal Iba-1 + immune cells in ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. IF of anti-C4 mAb (green), anti-Iba1 mAb (red, a microglia and monocyte/macrophage marker ). Staining cell nuclei (blue) is shown in the merged images (right column). Images were acquired using the Leica STELLARIS 8 FALCON Confocal Microscope, 40x objective

    Techniques Used: Sequencing, Quantitative RT-PCR, Western Blot, Staining, Marker, Microscopy

    hc3 ki rats  (Hycult Biotech)


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    Hycult Biotech hc3 ki rats
    Hc3 Ki Rats, supplied by Hycult Biotech, 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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    hc3 ki rats  (Hycult Biotech)


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    Hycult Biotech hc3 ki rats
    Hc3 Ki Rats, supplied by Hycult Biotech, 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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    hc3 ki rats  (Hycult Biotech)


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    Hycult Biotech hc3 ki rats
    Hc3 Ki Rats, supplied by Hycult Biotech, 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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    hc3 ki rats  (Hycult Biotech)


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    Hycult Biotech hc3 ki rats
    Hc3 Ki Rats, supplied by Hycult Biotech, 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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    rat anti c3  (Hycult Biotech)


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    Hycult Biotech rat anti c3
    Rat Anti C3, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rat anti atp7a  (Hycult Biotech)


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    Hycult Biotech rat anti atp7a
    Cu transporter <t>Atp7a</t> upregulates and relocates during adipogenesis. (A) Immunoblot of Atp7a and Atp7b in differentiated 3T3-L1 adipocytes on day 8. (B) Protein levels of Atp7a in pre-differentiated (pre-Adi), differentiated adipocytes (Adip). (C) Relative mRNA levels of Atp7a , normalized to non-differentiated cells (ND) (n = 3). (D) Immunoblot of Atp7a in 3T3-L1 cells in early differentiation day 3 after adipogenic stimuli and (E) associated quantifications. (F) Relative mRNA levels of Ctr1 in pre-adipocytes, on day 0, day 2, day 4, day 6, and day 8 after adipogenic stimuli, normalized to the levels in preadipocytes (n = 3). (G) Immunostaining of Atp7a (green) and TGN marker (syntaxin 6, red) in pre-adipocytes, on day 0, day 3, day 5, and day 8 after adipogenic stimuli. All values represent the mean ± SEM; One-way ANOVA was used to analyze the data in C and F, Student t-test was used for E. ∗ – p < 0.05, ∗∗∗ – p < 0.001.
    Rat Anti Atp7a, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90/100 stars

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    1) Product Images from "ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis"

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2024.101872

    Cu transporter Atp7a upregulates and relocates during adipogenesis. (A) Immunoblot of Atp7a and Atp7b in differentiated 3T3-L1 adipocytes on day 8. (B) Protein levels of Atp7a in pre-differentiated (pre-Adi), differentiated adipocytes (Adip). (C) Relative mRNA levels of Atp7a , normalized to non-differentiated cells (ND) (n = 3). (D) Immunoblot of Atp7a in 3T3-L1 cells in early differentiation day 3 after adipogenic stimuli and (E) associated quantifications. (F) Relative mRNA levels of Ctr1 in pre-adipocytes, on day 0, day 2, day 4, day 6, and day 8 after adipogenic stimuli, normalized to the levels in preadipocytes (n = 3). (G) Immunostaining of Atp7a (green) and TGN marker (syntaxin 6, red) in pre-adipocytes, on day 0, day 3, day 5, and day 8 after adipogenic stimuli. All values represent the mean ± SEM; One-way ANOVA was used to analyze the data in C and F, Student t-test was used for E. ∗ – p < 0.05, ∗∗∗ – p < 0.001.
    Figure Legend Snippet: Cu transporter Atp7a upregulates and relocates during adipogenesis. (A) Immunoblot of Atp7a and Atp7b in differentiated 3T3-L1 adipocytes on day 8. (B) Protein levels of Atp7a in pre-differentiated (pre-Adi), differentiated adipocytes (Adip). (C) Relative mRNA levels of Atp7a , normalized to non-differentiated cells (ND) (n = 3). (D) Immunoblot of Atp7a in 3T3-L1 cells in early differentiation day 3 after adipogenic stimuli and (E) associated quantifications. (F) Relative mRNA levels of Ctr1 in pre-adipocytes, on day 0, day 2, day 4, day 6, and day 8 after adipogenic stimuli, normalized to the levels in preadipocytes (n = 3). (G) Immunostaining of Atp7a (green) and TGN marker (syntaxin 6, red) in pre-adipocytes, on day 0, day 3, day 5, and day 8 after adipogenic stimuli. All values represent the mean ± SEM; One-way ANOVA was used to analyze the data in C and F, Student t-test was used for E. ∗ – p < 0.05, ∗∗∗ – p < 0.001.

    Techniques Used: Western Blot, Immunostaining, Marker

    Atp7a inactivation is associated with changes in Cu distribution, cell morphology and inhibition of adipogenesis. (A) X-ray fluorescence imaging and quantification of Cu in control (WT) and Atp7a −/− pre-adipocytes. Signal of phosphate (P) serves as an abundant internal control and identification of the nucleus in cells. (B) Representative images of cell morphology and oil red O stained differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (C) Relative triglyceride content in differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (D) Relative mRNA levels of C/EBPβ and (E) C/EBPδ in WT and Atp7a −/− cells during the first 24 h following stimulation of adipogenesis, normalized to WT cells at time 0 (n = 3). (F) Relative mRNA levels of PPARγ and C/EBPα in Atp7a −/− cells on day 0 and day 3, normalized to WT cells on day 0 (n = 3). All values represent the mean ± SEM. Student t -est was used for C and two-way ANOVA have been used for A, D, E and F; ns – p > 0.05, ∗ – p < 0.05, ∗∗ – p < 0.01, ∗∗∗∗ – p < 0.0001.
    Figure Legend Snippet: Atp7a inactivation is associated with changes in Cu distribution, cell morphology and inhibition of adipogenesis. (A) X-ray fluorescence imaging and quantification of Cu in control (WT) and Atp7a −/− pre-adipocytes. Signal of phosphate (P) serves as an abundant internal control and identification of the nucleus in cells. (B) Representative images of cell morphology and oil red O stained differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (C) Relative triglyceride content in differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (D) Relative mRNA levels of C/EBPβ and (E) C/EBPδ in WT and Atp7a −/− cells during the first 24 h following stimulation of adipogenesis, normalized to WT cells at time 0 (n = 3). (F) Relative mRNA levels of PPARγ and C/EBPα in Atp7a −/− cells on day 0 and day 3, normalized to WT cells on day 0 (n = 3). All values represent the mean ± SEM. Student t -est was used for C and two-way ANOVA have been used for A, D, E and F; ns – p > 0.05, ∗ – p < 0.05, ∗∗ – p < 0.01, ∗∗∗∗ – p < 0.0001.

    Techniques Used: Inhibition, Fluorescence, Imaging, Staining

    Analysis of WT and Atp7a −/− 3T3-L1 cells proteomes during early differentiation. (A) Schematic overview of TMT labeling mass spectrometry. (B) Summary of processes primarily changed in 3T3-L1 cells during day0-day3 transition: orange – upregulated, blue down-regulated. (C) Major functional differences between the 3T3-L1 and Atp7a −/− cells on day 3. (D) Immunostaining of α-tubulin in WT and Atp7a −/− 3T3-L1 preadipocytes. (E) Heatmap of highly changed proteins in WT 3T3-L1 during early adipogenesis stage, the cutoff used is 4-fold change. Clustering function in Heatmap.2 package. The box area is the list indicates the least down-regulated genes in Atp7a −/− cells compared to control adipocytes; β-catenin is among these least down-regulated proteins. (F) Table of log 2 fold change in abundance for proteins involved in Wnt/β-catenin pathway during D0-D3 transition.
    Figure Legend Snippet: Analysis of WT and Atp7a −/− 3T3-L1 cells proteomes during early differentiation. (A) Schematic overview of TMT labeling mass spectrometry. (B) Summary of processes primarily changed in 3T3-L1 cells during day0-day3 transition: orange – upregulated, blue down-regulated. (C) Major functional differences between the 3T3-L1 and Atp7a −/− cells on day 3. (D) Immunostaining of α-tubulin in WT and Atp7a −/− 3T3-L1 preadipocytes. (E) Heatmap of highly changed proteins in WT 3T3-L1 during early adipogenesis stage, the cutoff used is 4-fold change. Clustering function in Heatmap.2 package. The box area is the list indicates the least down-regulated genes in Atp7a −/− cells compared to control adipocytes; β-catenin is among these least down-regulated proteins. (F) Table of log 2 fold change in abundance for proteins involved in Wnt/β-catenin pathway during D0-D3 transition.

    Techniques Used: Labeling, Mass Spectrometry, Functional Assay, Immunostaining

    Wnt/β-catenin signaling persists in Cu overloaded cells during adipogensis . (A) Relative mRNA levels of C/EBPα , Wnt10b , Wnt10a and Wnt6 in Atp7a −/− 3T3-L1 normalized to WT cells on day 3 (n = 3). (B) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on day 0 and day 3, α-tubulin was used as internal control. (C) Immunoblot of β-catenin, phosphorylated-β-catenin, and Gsk3 in WT and Atp7a −/− 3T3-L1 cells on day 3 and quantification for (D) phosphorylation levels of β-catenin. (E) Immunostaining of β-catenin (green) and DAPI (blue) in WT and Atp7a −/− 3T3-L1 cells on day 3 after differentiation stimuli. (F) Relative triglyceride levels in differentiated Atp7a −/− cells (day 8) in either basal medium or medium containing 5 μM or 10 μM of the β-catenin inhibitor FH535 from day0 to day 3 during adipogenesis (n = 3). All values represent the mean ± SEM. Student t test was used for D, One-way ANOVA was used for F and two-way ANOVA was used for A. ns – p > 0.05 ∗ – p < 0.05, ∗∗∗ – p < 0.001.
    Figure Legend Snippet: Wnt/β-catenin signaling persists in Cu overloaded cells during adipogensis . (A) Relative mRNA levels of C/EBPα , Wnt10b , Wnt10a and Wnt6 in Atp7a −/− 3T3-L1 normalized to WT cells on day 3 (n = 3). (B) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on day 0 and day 3, α-tubulin was used as internal control. (C) Immunoblot of β-catenin, phosphorylated-β-catenin, and Gsk3 in WT and Atp7a −/− 3T3-L1 cells on day 3 and quantification for (D) phosphorylation levels of β-catenin. (E) Immunostaining of β-catenin (green) and DAPI (blue) in WT and Atp7a −/− 3T3-L1 cells on day 3 after differentiation stimuli. (F) Relative triglyceride levels in differentiated Atp7a −/− cells (day 8) in either basal medium or medium containing 5 μM or 10 μM of the β-catenin inhibitor FH535 from day0 to day 3 during adipogenesis (n = 3). All values represent the mean ± SEM. Student t test was used for D, One-way ANOVA was used for F and two-way ANOVA was used for A. ns – p > 0.05 ∗ – p < 0.05, ∗∗∗ – p < 0.001.

    Techniques Used: Western Blot, Immunostaining

    Differen t redox environment in control (WT) and Atp7a −/− cells contributes to different β-catenin stability. (A) Ratiometric representation and (B) quantification of Grx-roGFP signal in cytosol of non-differentiated WT cells (WT_ND) and WT cells on day 3 after differentiation stimuli (n = 38). (C) Ratiometric representation and (D) quantification of Grx-roGFP signal in cytosol in control (WT) and Atp7a −/− 3T3-L1 cells (n = 24). (E) Western blot of β-catenin on day 3 after stimulation treated with different concentration of NAC, β-actin was used as internal control. All values represent the mean ± SEM. Student t-test was used for B and D. ns – p > 0.05 ∗ – p < 0.05.
    Figure Legend Snippet: Differen t redox environment in control (WT) and Atp7a −/− cells contributes to different β-catenin stability. (A) Ratiometric representation and (B) quantification of Grx-roGFP signal in cytosol of non-differentiated WT cells (WT_ND) and WT cells on day 3 after differentiation stimuli (n = 38). (C) Ratiometric representation and (D) quantification of Grx-roGFP signal in cytosol in control (WT) and Atp7a −/− 3T3-L1 cells (n = 24). (E) Western blot of β-catenin on day 3 after stimulation treated with different concentration of NAC, β-actin was used as internal control. All values represent the mean ± SEM. Student t-test was used for B and D. ns – p > 0.05 ∗ – p < 0.05.

    Techniques Used: Western Blot, Concentration Assay

    Decrease of Cu normalizes β-catenin degradation, localization, and adipogenesis. (A) Triglyceride levels in differentiated Atp7a −/− cells in basal medium following expression of WT ATP7B. (B) Immunostaining of ATP7B-GFP (red) and β-catenin (green) in Atp7a −/− 3T3-L1 cells on day 3. (C) Immunoblot of ATP7B and β-catenin in Atp7a −/− 3T3-L1 cells on D3 which infected with wild type ATP7B-GFP and DA mutant ATP7B-GFP. (D) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on D3 incubated with 50 μM or 100 μM Cu chelator BCS since 2 days before and during differentiation. (E) Western blot and (F) densitometry (right) illustrate elevation of β-catenin in Cu overloaded livers of Atp7b −/− mice (n = 5) compared to WT livers. All values represent the mean ± SEM. Student t-test was used for A and F, ∗∗∗ – p < 0.001.
    Figure Legend Snippet: Decrease of Cu normalizes β-catenin degradation, localization, and adipogenesis. (A) Triglyceride levels in differentiated Atp7a −/− cells in basal medium following expression of WT ATP7B. (B) Immunostaining of ATP7B-GFP (red) and β-catenin (green) in Atp7a −/− 3T3-L1 cells on day 3. (C) Immunoblot of ATP7B and β-catenin in Atp7a −/− 3T3-L1 cells on D3 which infected with wild type ATP7B-GFP and DA mutant ATP7B-GFP. (D) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on D3 incubated with 50 μM or 100 μM Cu chelator BCS since 2 days before and during differentiation. (E) Western blot and (F) densitometry (right) illustrate elevation of β-catenin in Cu overloaded livers of Atp7b −/− mice (n = 5) compared to WT livers. All values represent the mean ± SEM. Student t-test was used for A and F, ∗∗∗ – p < 0.001.

    Techniques Used: Expressing, Immunostaining, Western Blot, Infection, Mutagenesis, Incubation

    anti rat cd55 antibody  (Hycult Biotech)


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    Hycult Biotech anti rat cd55 antibody
    ( a ) Flow cytometry image gallery: Constitutive HO-1 and <t>CD55</t> expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.
    Anti Rat Cd55 Antibody, supplied by Hycult Biotech, 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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    anti rat cd55 antibody - by Bioz Stars, 2024-04
    86/100 stars

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    1) Product Images from "Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes"

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    Journal: Biomedicines

    doi: 10.3390/biomedicines11123297

    ( a ) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.
    Figure Legend Snippet: ( a ) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.

    Techniques Used: Flow Cytometry, Expressing, Cell Culture, Staining, Imaging, Cytometry, Luminex, Incubation

    Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.
    Figure Legend Snippet: Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    ( a ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. ( b ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.
    Figure Legend Snippet: ( a ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. ( b ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.

    Techniques Used: Transfection, Negative Control, Expressing, Concentration Assay

    Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.
    Figure Legend Snippet: Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.

    Techniques Used: Transfection

    Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures ( panel A ), and in CD55 mRNA levels assessed by RT-PCR ( panel B ) in podocytes transfected with HO-1 siRNA. In the duplicate experiment ( panel A ), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA ( B ).
    Figure Legend Snippet: Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures ( panel A ), and in CD55 mRNA levels assessed by RT-PCR ( panel B ) in podocytes transfected with HO-1 siRNA. In the duplicate experiment ( panel A ), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA ( B ).

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Concentration Assay

    Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression , it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.
    Figure Legend Snippet: Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression , it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.

    Techniques Used: Over Expression, Isolation, Cell Culture, Expressing

    anti rat cd55 antibody  (Hycult Biotech)


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    Hycult Biotech anti rat cd55 antibody
    ( a ) Flow cytometry image gallery: Constitutive HO-1 and <t>CD55</t> expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.
    Anti Rat Cd55 Antibody, supplied by Hycult Biotech, 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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    1) Product Images from "Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes"

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    Journal: Biomedicines

    doi: 10.3390/biomedicines11123297

    ( a ) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.
    Figure Legend Snippet: ( a ) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.

    Techniques Used: Flow Cytometry, Expressing, Cell Culture, Staining, Imaging, Cytometry, Luminex, Incubation

    Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.
    Figure Legend Snippet: Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    ( a ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. ( b ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.
    Figure Legend Snippet: ( a ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. ( b ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.

    Techniques Used: Transfection, Negative Control, Expressing, Concentration Assay

    Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.
    Figure Legend Snippet: Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.

    Techniques Used: Transfection

    Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures ( panel A ), and in CD55 mRNA levels assessed by RT-PCR ( panel B ) in podocytes transfected with HO-1 siRNA. In the duplicate experiment ( panel A ), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA ( B ).
    Figure Legend Snippet: Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures ( panel A ), and in CD55 mRNA levels assessed by RT-PCR ( panel B ) in podocytes transfected with HO-1 siRNA. In the duplicate experiment ( panel A ), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA ( B ).

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Concentration Assay

    Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression , it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.
    Figure Legend Snippet: Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression , it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.

    Techniques Used: Over Expression, Isolation, Cell Culture, Expressing

    anti rat cd55 antibody  (Hycult Biotech)


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    Hycult Biotech anti rat cd55 antibody
    Anti Rat Cd55 Antibody, supplied by Hycult Biotech, 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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    ( a ) Flow cytometry image gallery: Constitutive HO-1 and <t>CD55</t> expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.
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    C4 protein localization after retinal detachment for sequencing validation. ( A ) QRT-PCR analysis of C4b mRNA levels in naïve and detached RPE at 1 dprd and 7 dprd. ( B ) Representative immunoblot of complement C4 in the soluble fluid fraction from C57BL6/J mouse eyes under naïve, contralateral (fellow at 7 dprd), and detached (7 dprd) conditions with protein samples separated under non-reducing conditions. Blots are representative of 3 independent animals in each condition. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Dunnett’s post hoc test. * p < 0.05; **** p < 0.0001. ( C ) IF analysis of ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. Cell nuclei staining (blue), and IF of anti-RPE65 mAb (green) and anti-C4 serum (red) are shown. ( D ) IF analysis of C4 protein in retinal and subretinal Iba-1 + immune cells in ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. IF of anti-C4 mAb (green), anti-Iba1 mAb (red, a microglia and monocyte/macrophage marker ). Staining cell nuclei (blue) is shown in the merged images (right column). Images were acquired using the Leica STELLARIS 8 FALCON Confocal Microscope, 40x objective

    Journal: Journal of Neuroinflammation

    Article Title: The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment

    doi: 10.1186/s12974-024-03062-2

    Figure Lengend Snippet: C4 protein localization after retinal detachment for sequencing validation. ( A ) QRT-PCR analysis of C4b mRNA levels in naïve and detached RPE at 1 dprd and 7 dprd. ( B ) Representative immunoblot of complement C4 in the soluble fluid fraction from C57BL6/J mouse eyes under naïve, contralateral (fellow at 7 dprd), and detached (7 dprd) conditions with protein samples separated under non-reducing conditions. Blots are representative of 3 independent animals in each condition. Bar graphs represent mean ± SEM. Statistical analysis was performed using one-way ANOVA with repeated measures followed by Dunnett’s post hoc test. * p < 0.05; **** p < 0.0001. ( C ) IF analysis of ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. Cell nuclei staining (blue), and IF of anti-RPE65 mAb (green) and anti-C4 serum (red) are shown. ( D ) IF analysis of C4 protein in retinal and subretinal Iba-1 + immune cells in ocular sections from C57BL6/J mice under naïve, 1 dprd and 7 dprd conditions. IF of anti-C4 mAb (green), anti-Iba1 mAb (red, a microglia and monocyte/macrophage marker ). Staining cell nuclei (blue) is shown in the merged images (right column). Images were acquired using the Leica STELLARIS 8 FALCON Confocal Microscope, 40x objective

    Article Snippet: Eye sections were subjected to blocking in TBS with 10% Donkey serum and 0.3% Triton X-100 (TBST) at RT for one hour, and incubated overnight at 4 °C with a mixture of primary antibodies in blocking buffer, including: Anti-GSTO1 rabbit pAb (dilution 1:100, Proteintech), mouse anti-RPE65 mAb (clone 8B11.37, dilution 1:250, a gift from Dr. Debra Thompson, University of Michigan Medicine Kellogg Eye Center), goat anti-mouse LCN2/NGAL pAb (1:1000; catalog# AF1857, R&D Systems), goat anti-serum raised against human C4 (1:25, Complement Technology, Inc.), rat anti-C4 mAb (clone 16D2, 1:1000, Hycult Biotech) and rabbit anti-AIF-1/Iba1 pAb (1:100, Novus Biologicals, NB100-1028).

    Techniques: Sequencing, Quantitative RT-PCR, Western Blot, Staining, Marker, Microscopy

    Cu transporter Atp7a upregulates and relocates during adipogenesis. (A) Immunoblot of Atp7a and Atp7b in differentiated 3T3-L1 adipocytes on day 8. (B) Protein levels of Atp7a in pre-differentiated (pre-Adi), differentiated adipocytes (Adip). (C) Relative mRNA levels of Atp7a , normalized to non-differentiated cells (ND) (n = 3). (D) Immunoblot of Atp7a in 3T3-L1 cells in early differentiation day 3 after adipogenic stimuli and (E) associated quantifications. (F) Relative mRNA levels of Ctr1 in pre-adipocytes, on day 0, day 2, day 4, day 6, and day 8 after adipogenic stimuli, normalized to the levels in preadipocytes (n = 3). (G) Immunostaining of Atp7a (green) and TGN marker (syntaxin 6, red) in pre-adipocytes, on day 0, day 3, day 5, and day 8 after adipogenic stimuli. All values represent the mean ± SEM; One-way ANOVA was used to analyze the data in C and F, Student t-test was used for E. ∗ – p < 0.05, ∗∗∗ – p < 0.001.

    Journal: Molecular Metabolism

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    doi: 10.1016/j.molmet.2024.101872

    Figure Lengend Snippet: Cu transporter Atp7a upregulates and relocates during adipogenesis. (A) Immunoblot of Atp7a and Atp7b in differentiated 3T3-L1 adipocytes on day 8. (B) Protein levels of Atp7a in pre-differentiated (pre-Adi), differentiated adipocytes (Adip). (C) Relative mRNA levels of Atp7a , normalized to non-differentiated cells (ND) (n = 3). (D) Immunoblot of Atp7a in 3T3-L1 cells in early differentiation day 3 after adipogenic stimuli and (E) associated quantifications. (F) Relative mRNA levels of Ctr1 in pre-adipocytes, on day 0, day 2, day 4, day 6, and day 8 after adipogenic stimuli, normalized to the levels in preadipocytes (n = 3). (G) Immunostaining of Atp7a (green) and TGN marker (syntaxin 6, red) in pre-adipocytes, on day 0, day 3, day 5, and day 8 after adipogenic stimuli. All values represent the mean ± SEM; One-way ANOVA was used to analyze the data in C and F, Student t-test was used for E. ∗ – p < 0.05, ∗∗∗ – p < 0.001.

    Article Snippet: Primary antibodies used in immunoblotting were: rabbit monoclonal anti-mouse and rat anti-ATP7A (Hycult biotech), mouse anti-Syntaxin-6 (BD Transduction Laboratories, 610,636), rabbit polyclonal anti-beta catenin (ab16051), mouse anti-GM-130 (BD Biosciences, 610,822).

    Techniques: Western Blot, Immunostaining, Marker

    Atp7a inactivation is associated with changes in Cu distribution, cell morphology and inhibition of adipogenesis. (A) X-ray fluorescence imaging and quantification of Cu in control (WT) and Atp7a −/− pre-adipocytes. Signal of phosphate (P) serves as an abundant internal control and identification of the nucleus in cells. (B) Representative images of cell morphology and oil red O stained differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (C) Relative triglyceride content in differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (D) Relative mRNA levels of C/EBPβ and (E) C/EBPδ in WT and Atp7a −/− cells during the first 24 h following stimulation of adipogenesis, normalized to WT cells at time 0 (n = 3). (F) Relative mRNA levels of PPARγ and C/EBPα in Atp7a −/− cells on day 0 and day 3, normalized to WT cells on day 0 (n = 3). All values represent the mean ± SEM. Student t -est was used for C and two-way ANOVA have been used for A, D, E and F; ns – p > 0.05, ∗ – p < 0.05, ∗∗ – p < 0.01, ∗∗∗∗ – p < 0.0001.

    Journal: Molecular Metabolism

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    doi: 10.1016/j.molmet.2024.101872

    Figure Lengend Snippet: Atp7a inactivation is associated with changes in Cu distribution, cell morphology and inhibition of adipogenesis. (A) X-ray fluorescence imaging and quantification of Cu in control (WT) and Atp7a −/− pre-adipocytes. Signal of phosphate (P) serves as an abundant internal control and identification of the nucleus in cells. (B) Representative images of cell morphology and oil red O stained differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (C) Relative triglyceride content in differentiated WT and Atp7a −/− 3T3-L1 adipocytes on day 8 (n = 3). (D) Relative mRNA levels of C/EBPβ and (E) C/EBPδ in WT and Atp7a −/− cells during the first 24 h following stimulation of adipogenesis, normalized to WT cells at time 0 (n = 3). (F) Relative mRNA levels of PPARγ and C/EBPα in Atp7a −/− cells on day 0 and day 3, normalized to WT cells on day 0 (n = 3). All values represent the mean ± SEM. Student t -est was used for C and two-way ANOVA have been used for A, D, E and F; ns – p > 0.05, ∗ – p < 0.05, ∗∗ – p < 0.01, ∗∗∗∗ – p < 0.0001.

    Article Snippet: Primary antibodies used in immunoblotting were: rabbit monoclonal anti-mouse and rat anti-ATP7A (Hycult biotech), mouse anti-Syntaxin-6 (BD Transduction Laboratories, 610,636), rabbit polyclonal anti-beta catenin (ab16051), mouse anti-GM-130 (BD Biosciences, 610,822).

    Techniques: Inhibition, Fluorescence, Imaging, Staining

    Analysis of WT and Atp7a −/− 3T3-L1 cells proteomes during early differentiation. (A) Schematic overview of TMT labeling mass spectrometry. (B) Summary of processes primarily changed in 3T3-L1 cells during day0-day3 transition: orange – upregulated, blue down-regulated. (C) Major functional differences between the 3T3-L1 and Atp7a −/− cells on day 3. (D) Immunostaining of α-tubulin in WT and Atp7a −/− 3T3-L1 preadipocytes. (E) Heatmap of highly changed proteins in WT 3T3-L1 during early adipogenesis stage, the cutoff used is 4-fold change. Clustering function in Heatmap.2 package. The box area is the list indicates the least down-regulated genes in Atp7a −/− cells compared to control adipocytes; β-catenin is among these least down-regulated proteins. (F) Table of log 2 fold change in abundance for proteins involved in Wnt/β-catenin pathway during D0-D3 transition.

    Journal: Molecular Metabolism

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    doi: 10.1016/j.molmet.2024.101872

    Figure Lengend Snippet: Analysis of WT and Atp7a −/− 3T3-L1 cells proteomes during early differentiation. (A) Schematic overview of TMT labeling mass spectrometry. (B) Summary of processes primarily changed in 3T3-L1 cells during day0-day3 transition: orange – upregulated, blue down-regulated. (C) Major functional differences between the 3T3-L1 and Atp7a −/− cells on day 3. (D) Immunostaining of α-tubulin in WT and Atp7a −/− 3T3-L1 preadipocytes. (E) Heatmap of highly changed proteins in WT 3T3-L1 during early adipogenesis stage, the cutoff used is 4-fold change. Clustering function in Heatmap.2 package. The box area is the list indicates the least down-regulated genes in Atp7a −/− cells compared to control adipocytes; β-catenin is among these least down-regulated proteins. (F) Table of log 2 fold change in abundance for proteins involved in Wnt/β-catenin pathway during D0-D3 transition.

    Article Snippet: Primary antibodies used in immunoblotting were: rabbit monoclonal anti-mouse and rat anti-ATP7A (Hycult biotech), mouse anti-Syntaxin-6 (BD Transduction Laboratories, 610,636), rabbit polyclonal anti-beta catenin (ab16051), mouse anti-GM-130 (BD Biosciences, 610,822).

    Techniques: Labeling, Mass Spectrometry, Functional Assay, Immunostaining

    Wnt/β-catenin signaling persists in Cu overloaded cells during adipogensis . (A) Relative mRNA levels of C/EBPα , Wnt10b , Wnt10a and Wnt6 in Atp7a −/− 3T3-L1 normalized to WT cells on day 3 (n = 3). (B) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on day 0 and day 3, α-tubulin was used as internal control. (C) Immunoblot of β-catenin, phosphorylated-β-catenin, and Gsk3 in WT and Atp7a −/− 3T3-L1 cells on day 3 and quantification for (D) phosphorylation levels of β-catenin. (E) Immunostaining of β-catenin (green) and DAPI (blue) in WT and Atp7a −/− 3T3-L1 cells on day 3 after differentiation stimuli. (F) Relative triglyceride levels in differentiated Atp7a −/− cells (day 8) in either basal medium or medium containing 5 μM or 10 μM of the β-catenin inhibitor FH535 from day0 to day 3 during adipogenesis (n = 3). All values represent the mean ± SEM. Student t test was used for D, One-way ANOVA was used for F and two-way ANOVA was used for A. ns – p > 0.05 ∗ – p < 0.05, ∗∗∗ – p < 0.001.

    Journal: Molecular Metabolism

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    doi: 10.1016/j.molmet.2024.101872

    Figure Lengend Snippet: Wnt/β-catenin signaling persists in Cu overloaded cells during adipogensis . (A) Relative mRNA levels of C/EBPα , Wnt10b , Wnt10a and Wnt6 in Atp7a −/− 3T3-L1 normalized to WT cells on day 3 (n = 3). (B) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on day 0 and day 3, α-tubulin was used as internal control. (C) Immunoblot of β-catenin, phosphorylated-β-catenin, and Gsk3 in WT and Atp7a −/− 3T3-L1 cells on day 3 and quantification for (D) phosphorylation levels of β-catenin. (E) Immunostaining of β-catenin (green) and DAPI (blue) in WT and Atp7a −/− 3T3-L1 cells on day 3 after differentiation stimuli. (F) Relative triglyceride levels in differentiated Atp7a −/− cells (day 8) in either basal medium or medium containing 5 μM or 10 μM of the β-catenin inhibitor FH535 from day0 to day 3 during adipogenesis (n = 3). All values represent the mean ± SEM. Student t test was used for D, One-way ANOVA was used for F and two-way ANOVA was used for A. ns – p > 0.05 ∗ – p < 0.05, ∗∗∗ – p < 0.001.

    Article Snippet: Primary antibodies used in immunoblotting were: rabbit monoclonal anti-mouse and rat anti-ATP7A (Hycult biotech), mouse anti-Syntaxin-6 (BD Transduction Laboratories, 610,636), rabbit polyclonal anti-beta catenin (ab16051), mouse anti-GM-130 (BD Biosciences, 610,822).

    Techniques: Western Blot, Immunostaining

    Differen t redox environment in control (WT) and Atp7a −/− cells contributes to different β-catenin stability. (A) Ratiometric representation and (B) quantification of Grx-roGFP signal in cytosol of non-differentiated WT cells (WT_ND) and WT cells on day 3 after differentiation stimuli (n = 38). (C) Ratiometric representation and (D) quantification of Grx-roGFP signal in cytosol in control (WT) and Atp7a −/− 3T3-L1 cells (n = 24). (E) Western blot of β-catenin on day 3 after stimulation treated with different concentration of NAC, β-actin was used as internal control. All values represent the mean ± SEM. Student t-test was used for B and D. ns – p > 0.05 ∗ – p < 0.05.

    Journal: Molecular Metabolism

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    doi: 10.1016/j.molmet.2024.101872

    Figure Lengend Snippet: Differen t redox environment in control (WT) and Atp7a −/− cells contributes to different β-catenin stability. (A) Ratiometric representation and (B) quantification of Grx-roGFP signal in cytosol of non-differentiated WT cells (WT_ND) and WT cells on day 3 after differentiation stimuli (n = 38). (C) Ratiometric representation and (D) quantification of Grx-roGFP signal in cytosol in control (WT) and Atp7a −/− 3T3-L1 cells (n = 24). (E) Western blot of β-catenin on day 3 after stimulation treated with different concentration of NAC, β-actin was used as internal control. All values represent the mean ± SEM. Student t-test was used for B and D. ns – p > 0.05 ∗ – p < 0.05.

    Article Snippet: Primary antibodies used in immunoblotting were: rabbit monoclonal anti-mouse and rat anti-ATP7A (Hycult biotech), mouse anti-Syntaxin-6 (BD Transduction Laboratories, 610,636), rabbit polyclonal anti-beta catenin (ab16051), mouse anti-GM-130 (BD Biosciences, 610,822).

    Techniques: Western Blot, Concentration Assay

    Decrease of Cu normalizes β-catenin degradation, localization, and adipogenesis. (A) Triglyceride levels in differentiated Atp7a −/− cells in basal medium following expression of WT ATP7B. (B) Immunostaining of ATP7B-GFP (red) and β-catenin (green) in Atp7a −/− 3T3-L1 cells on day 3. (C) Immunoblot of ATP7B and β-catenin in Atp7a −/− 3T3-L1 cells on D3 which infected with wild type ATP7B-GFP and DA mutant ATP7B-GFP. (D) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on D3 incubated with 50 μM or 100 μM Cu chelator BCS since 2 days before and during differentiation. (E) Western blot and (F) densitometry (right) illustrate elevation of β-catenin in Cu overloaded livers of Atp7b −/− mice (n = 5) compared to WT livers. All values represent the mean ± SEM. Student t-test was used for A and F, ∗∗∗ – p < 0.001.

    Journal: Molecular Metabolism

    Article Title: ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

    doi: 10.1016/j.molmet.2024.101872

    Figure Lengend Snippet: Decrease of Cu normalizes β-catenin degradation, localization, and adipogenesis. (A) Triglyceride levels in differentiated Atp7a −/− cells in basal medium following expression of WT ATP7B. (B) Immunostaining of ATP7B-GFP (red) and β-catenin (green) in Atp7a −/− 3T3-L1 cells on day 3. (C) Immunoblot of ATP7B and β-catenin in Atp7a −/− 3T3-L1 cells on D3 which infected with wild type ATP7B-GFP and DA mutant ATP7B-GFP. (D) Immunoblot of β-catenin in WT and Atp7a −/− 3T3-L1 cells on D3 incubated with 50 μM or 100 μM Cu chelator BCS since 2 days before and during differentiation. (E) Western blot and (F) densitometry (right) illustrate elevation of β-catenin in Cu overloaded livers of Atp7b −/− mice (n = 5) compared to WT livers. All values represent the mean ± SEM. Student t-test was used for A and F, ∗∗∗ – p < 0.001.

    Article Snippet: Primary antibodies used in immunoblotting were: rabbit monoclonal anti-mouse and rat anti-ATP7A (Hycult biotech), mouse anti-Syntaxin-6 (BD Transduction Laboratories, 610,636), rabbit polyclonal anti-beta catenin (ab16051), mouse anti-GM-130 (BD Biosciences, 610,822).

    Techniques: Expressing, Immunostaining, Western Blot, Infection, Mutagenesis, Incubation

    ( a ) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.

    Journal: Biomedicines

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    doi: 10.3390/biomedicines11123297

    Figure Lengend Snippet: ( a ) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). ( b ) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody ( left panel ) and of AF647 conjugated anti-rat HO-1 antibody ( right panel ) plotted against number of cells interrogated.

    Article Snippet: The following reagents , obtained from sources indicated , were used : Primary rat podocytes for culture and appropriate culture medium (Celprogen, Torrance, CA, USA), Iron Protoporhyrin IX (Heme, hemin, Tocris BioScience, Minneapolis, MN, USA), RIPA lysis/extraction buffer (Thermo Fisher Scientific, Grand Island, NY, USA), TRIzol reagent (Thermo Fisher Scientific), LDH-Glo cytotoxicity assay (Promega, Madison, WI, USA), Mycozap-Plus (Lonza, Durham, NC, USA), anti-rat CD32 FcγII receptor antibody (BD Pharmingen, San Jose, CA, USA), anti-rat Fx1A antibody (Avantor, Radnor, PA, USA), anti-rat HO-1 antibody (StressMarq, Vicroria, BC, Canada), anti-rat CD55 antibody (Hycult Biotech, Uden, The Netherlands), cDNA synthesis Kit (BioRad, Hercules, CA, USA), SsoAdvanced Universal SYBR Green Supermix (BioRad), TrypLE Express enzyme (Thermo Fisher Scientific), spin column-based RNA extraction kits (BioRad), siRNA duplexes targeting the rat hmox1 gene (Thermo Fisher Scientific), Lipofectamine RNAi-Max transfection reagent (Themo Fisher Scientific), SiRNA negative control (Thermo Fisher Scientific), Silencer Select GAPDH positive control (Thermo Fisher Scientific), fluorescent transfection control (Thermo Fisher Scientific), RT-qPCR primers for rat HO-1, CD55 and GAPDH (BioRad).

    Techniques: Flow Cytometry, Expressing, Cell Culture, Staining, Imaging, Cytometry, Luminex, Incubation

    Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.

    Journal: Biomedicines

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    doi: 10.3390/biomedicines11123297

    Figure Lengend Snippet: Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.

    Article Snippet: The following reagents , obtained from sources indicated , were used : Primary rat podocytes for culture and appropriate culture medium (Celprogen, Torrance, CA, USA), Iron Protoporhyrin IX (Heme, hemin, Tocris BioScience, Minneapolis, MN, USA), RIPA lysis/extraction buffer (Thermo Fisher Scientific, Grand Island, NY, USA), TRIzol reagent (Thermo Fisher Scientific), LDH-Glo cytotoxicity assay (Promega, Madison, WI, USA), Mycozap-Plus (Lonza, Durham, NC, USA), anti-rat CD32 FcγII receptor antibody (BD Pharmingen, San Jose, CA, USA), anti-rat Fx1A antibody (Avantor, Radnor, PA, USA), anti-rat HO-1 antibody (StressMarq, Vicroria, BC, Canada), anti-rat CD55 antibody (Hycult Biotech, Uden, The Netherlands), cDNA synthesis Kit (BioRad, Hercules, CA, USA), SsoAdvanced Universal SYBR Green Supermix (BioRad), TrypLE Express enzyme (Thermo Fisher Scientific), spin column-based RNA extraction kits (BioRad), siRNA duplexes targeting the rat hmox1 gene (Thermo Fisher Scientific), Lipofectamine RNAi-Max transfection reagent (Themo Fisher Scientific), SiRNA negative control (Thermo Fisher Scientific), Silencer Select GAPDH positive control (Thermo Fisher Scientific), fluorescent transfection control (Thermo Fisher Scientific), RT-qPCR primers for rat HO-1, CD55 and GAPDH (BioRad).

    Techniques: Expressing, Incubation, Quantitative RT-PCR

    ( a ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. ( b ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.

    Journal: Biomedicines

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    doi: 10.3390/biomedicines11123297

    Figure Lengend Snippet: ( a ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. ( b ) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.

    Article Snippet: The following reagents , obtained from sources indicated , were used : Primary rat podocytes for culture and appropriate culture medium (Celprogen, Torrance, CA, USA), Iron Protoporhyrin IX (Heme, hemin, Tocris BioScience, Minneapolis, MN, USA), RIPA lysis/extraction buffer (Thermo Fisher Scientific, Grand Island, NY, USA), TRIzol reagent (Thermo Fisher Scientific), LDH-Glo cytotoxicity assay (Promega, Madison, WI, USA), Mycozap-Plus (Lonza, Durham, NC, USA), anti-rat CD32 FcγII receptor antibody (BD Pharmingen, San Jose, CA, USA), anti-rat Fx1A antibody (Avantor, Radnor, PA, USA), anti-rat HO-1 antibody (StressMarq, Vicroria, BC, Canada), anti-rat CD55 antibody (Hycult Biotech, Uden, The Netherlands), cDNA synthesis Kit (BioRad, Hercules, CA, USA), SsoAdvanced Universal SYBR Green Supermix (BioRad), TrypLE Express enzyme (Thermo Fisher Scientific), spin column-based RNA extraction kits (BioRad), siRNA duplexes targeting the rat hmox1 gene (Thermo Fisher Scientific), Lipofectamine RNAi-Max transfection reagent (Themo Fisher Scientific), SiRNA negative control (Thermo Fisher Scientific), Silencer Select GAPDH positive control (Thermo Fisher Scientific), fluorescent transfection control (Thermo Fisher Scientific), RT-qPCR primers for rat HO-1, CD55 and GAPDH (BioRad).

    Techniques: Transfection, Negative Control, Expressing, Concentration Assay

    Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.

    Journal: Biomedicines

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    doi: 10.3390/biomedicines11123297

    Figure Lengend Snippet: Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.

    Article Snippet: The following reagents , obtained from sources indicated , were used : Primary rat podocytes for culture and appropriate culture medium (Celprogen, Torrance, CA, USA), Iron Protoporhyrin IX (Heme, hemin, Tocris BioScience, Minneapolis, MN, USA), RIPA lysis/extraction buffer (Thermo Fisher Scientific, Grand Island, NY, USA), TRIzol reagent (Thermo Fisher Scientific), LDH-Glo cytotoxicity assay (Promega, Madison, WI, USA), Mycozap-Plus (Lonza, Durham, NC, USA), anti-rat CD32 FcγII receptor antibody (BD Pharmingen, San Jose, CA, USA), anti-rat Fx1A antibody (Avantor, Radnor, PA, USA), anti-rat HO-1 antibody (StressMarq, Vicroria, BC, Canada), anti-rat CD55 antibody (Hycult Biotech, Uden, The Netherlands), cDNA synthesis Kit (BioRad, Hercules, CA, USA), SsoAdvanced Universal SYBR Green Supermix (BioRad), TrypLE Express enzyme (Thermo Fisher Scientific), spin column-based RNA extraction kits (BioRad), siRNA duplexes targeting the rat hmox1 gene (Thermo Fisher Scientific), Lipofectamine RNAi-Max transfection reagent (Themo Fisher Scientific), SiRNA negative control (Thermo Fisher Scientific), Silencer Select GAPDH positive control (Thermo Fisher Scientific), fluorescent transfection control (Thermo Fisher Scientific), RT-qPCR primers for rat HO-1, CD55 and GAPDH (BioRad).

    Techniques: Transfection

    Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures ( panel A ), and in CD55 mRNA levels assessed by RT-PCR ( panel B ) in podocytes transfected with HO-1 siRNA. In the duplicate experiment ( panel A ), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA ( B ).

    Journal: Biomedicines

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    doi: 10.3390/biomedicines11123297

    Figure Lengend Snippet: Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures ( panel A ), and in CD55 mRNA levels assessed by RT-PCR ( panel B ) in podocytes transfected with HO-1 siRNA. In the duplicate experiment ( panel A ), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA ( B ).

    Article Snippet: The following reagents , obtained from sources indicated , were used : Primary rat podocytes for culture and appropriate culture medium (Celprogen, Torrance, CA, USA), Iron Protoporhyrin IX (Heme, hemin, Tocris BioScience, Minneapolis, MN, USA), RIPA lysis/extraction buffer (Thermo Fisher Scientific, Grand Island, NY, USA), TRIzol reagent (Thermo Fisher Scientific), LDH-Glo cytotoxicity assay (Promega, Madison, WI, USA), Mycozap-Plus (Lonza, Durham, NC, USA), anti-rat CD32 FcγII receptor antibody (BD Pharmingen, San Jose, CA, USA), anti-rat Fx1A antibody (Avantor, Radnor, PA, USA), anti-rat HO-1 antibody (StressMarq, Vicroria, BC, Canada), anti-rat CD55 antibody (Hycult Biotech, Uden, The Netherlands), cDNA synthesis Kit (BioRad, Hercules, CA, USA), SsoAdvanced Universal SYBR Green Supermix (BioRad), TrypLE Express enzyme (Thermo Fisher Scientific), spin column-based RNA extraction kits (BioRad), siRNA duplexes targeting the rat hmox1 gene (Thermo Fisher Scientific), Lipofectamine RNAi-Max transfection reagent (Themo Fisher Scientific), SiRNA negative control (Thermo Fisher Scientific), Silencer Select GAPDH positive control (Thermo Fisher Scientific), fluorescent transfection control (Thermo Fisher Scientific), RT-qPCR primers for rat HO-1, CD55 and GAPDH (BioRad).

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Concentration Assay

    Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression , it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.

    Journal: Biomedicines

    Article Title: Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

    doi: 10.3390/biomedicines11123297

    Figure Lengend Snippet: Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression , it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.

    Article Snippet: The following reagents , obtained from sources indicated , were used : Primary rat podocytes for culture and appropriate culture medium (Celprogen, Torrance, CA, USA), Iron Protoporhyrin IX (Heme, hemin, Tocris BioScience, Minneapolis, MN, USA), RIPA lysis/extraction buffer (Thermo Fisher Scientific, Grand Island, NY, USA), TRIzol reagent (Thermo Fisher Scientific), LDH-Glo cytotoxicity assay (Promega, Madison, WI, USA), Mycozap-Plus (Lonza, Durham, NC, USA), anti-rat CD32 FcγII receptor antibody (BD Pharmingen, San Jose, CA, USA), anti-rat Fx1A antibody (Avantor, Radnor, PA, USA), anti-rat HO-1 antibody (StressMarq, Vicroria, BC, Canada), anti-rat CD55 antibody (Hycult Biotech, Uden, The Netherlands), cDNA synthesis Kit (BioRad, Hercules, CA, USA), SsoAdvanced Universal SYBR Green Supermix (BioRad), TrypLE Express enzyme (Thermo Fisher Scientific), spin column-based RNA extraction kits (BioRad), siRNA duplexes targeting the rat hmox1 gene (Thermo Fisher Scientific), Lipofectamine RNAi-Max transfection reagent (Themo Fisher Scientific), SiRNA negative control (Thermo Fisher Scientific), Silencer Select GAPDH positive control (Thermo Fisher Scientific), fluorescent transfection control (Thermo Fisher Scientific), RT-qPCR primers for rat HO-1, CD55 and GAPDH (BioRad).

    Techniques: Over Expression, Isolation, Cell Culture, Expressing