c3ar, human, mab 17  (Hycult Biotech)


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    Hycult Biotech c3ar, human, mab 17
    C3ar, Human, Mab 17, 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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    c3ar, human, mab 17  (Hycult Biotech)


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    Hycult Biotech c3ar, human, mab 17
    C3ar, Human, Mab 17, 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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    c3ar, human, mab 17  (Hycult Biotech)


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    Hycult Biotech c3ar, human, mab 17
    C3ar, Human, Mab 17, 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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    c3ar  (Hycult Biotech)


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    Hycult Biotech c3ar
    C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by <t>C3aR</t> antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.
    C3ar, 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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    1) Product Images from "C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway"

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28143-0

    C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by C3aR antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.
    Figure Legend Snippet: C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by C3aR antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.

    Techniques Used: Cell Culture

    C3aR antagonist prevents formation of basal deposits caused by C3a. SEM images of decellularized transwells show exposed basal deposits made by hfRPE cells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. C3a triggers the accumulation of deposits made of ECM fibers underneath the RPE. The abnormal deposition is prevented by the addition of C3aR. Scale bars a–c: 100 µm, d–i: 10 µm.
    Figure Legend Snippet: C3aR antagonist prevents formation of basal deposits caused by C3a. SEM images of decellularized transwells show exposed basal deposits made by hfRPE cells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. C3a triggers the accumulation of deposits made of ECM fibers underneath the RPE. The abnormal deposition is prevented by the addition of C3aR. Scale bars a–c: 100 µm, d–i: 10 µm.

    Techniques Used:

    C3a induces overexpression of C3aR in the basolateral membrane of the RPE. ( a ) Immunolabeling with C3aR antibodies of hfRPE cells treated with different doses of C3a (C3aR-ant) or C3a + C3aR antagonist (C3aR + ant) for 2 weeks. Z-stack was built from images taken every 0.5 µm with confocal microscope. 90° projections show that C3aR is expressed on the basal-lateral membrane of RPE. Scale bars 50 µm. ( b ) Average quantification of C3aR fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a + C3aR antagonist (C3aR + ant). (ANOVA, n = 4/C3aR-ant and n = 4/C3aR + ant treatment. Data represented as mean ± SEM. *p < 0.05, **p < 0.01). ( c ) mRNA levels of C3aR of hfRPE cells treated with different doses of C3a for 24 hours (ANOVA, n = 6. Data presented as mean ± SD. **p < 0.01).
    Figure Legend Snippet: C3a induces overexpression of C3aR in the basolateral membrane of the RPE. ( a ) Immunolabeling with C3aR antibodies of hfRPE cells treated with different doses of C3a (C3aR-ant) or C3a + C3aR antagonist (C3aR + ant) for 2 weeks. Z-stack was built from images taken every 0.5 µm with confocal microscope. 90° projections show that C3aR is expressed on the basal-lateral membrane of RPE. Scale bars 50 µm. ( b ) Average quantification of C3aR fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a + C3aR antagonist (C3aR + ant). (ANOVA, n = 4/C3aR-ant and n = 4/C3aR + ant treatment. Data represented as mean ± SEM. *p < 0.05, **p < 0.01). ( c ) mRNA levels of C3aR of hfRPE cells treated with different doses of C3a for 24 hours (ANOVA, n = 6. Data presented as mean ± SD. **p < 0.01).

    Techniques Used: Over Expression, Immunolabeling, Microscopy

    C3a causes specific deposition of Col IV and Col VI underneath the RPE that is prevented by C3aR antagonist. Immunolabeling with antibodies for ( a ) Col IV, ( c ) Col VI, ( e ) Col I, and ( g ) EFEMP1 of hfRPE cells treated with different doses of C3a or C3a + C3aR antagonist for 2 weeks. Images were taken with confocal. Orthogonal views show the basal deposition of the ECM proteins (left: apical, right: basal). Scale bars 50 µm. Average quantification of ( b ) Col IV, ( d ) Col VI, ( f ) Col I, and ( h ) EFEMP1 fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a plus C3aR antagonist (C3aR + ant) for 2 weeks. ( i ) Average quantification of immunostainings for ECM proteins after 4 weeks of treatment with C3a. (ANOVA. Data represented as mean ± SEM. n = 6/C3a dose *p < 0.05, **p < 0.01). ( j ) mRNA expression of COL4 and COL6 normalized to GAPDH after treatment with C3a for 2 weeks in the absence and presence (pattern bar) of C3aR antagonist (ANOVA. n = 3/−C3aR ant and n = 3/+ C3aR ant. Data represented as mean ± SD, **p < 0.01, ***p < 0.001, ****p < 0.0001).
    Figure Legend Snippet: C3a causes specific deposition of Col IV and Col VI underneath the RPE that is prevented by C3aR antagonist. Immunolabeling with antibodies for ( a ) Col IV, ( c ) Col VI, ( e ) Col I, and ( g ) EFEMP1 of hfRPE cells treated with different doses of C3a or C3a + C3aR antagonist for 2 weeks. Images were taken with confocal. Orthogonal views show the basal deposition of the ECM proteins (left: apical, right: basal). Scale bars 50 µm. Average quantification of ( b ) Col IV, ( d ) Col VI, ( f ) Col I, and ( h ) EFEMP1 fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a plus C3aR antagonist (C3aR + ant) for 2 weeks. ( i ) Average quantification of immunostainings for ECM proteins after 4 weeks of treatment with C3a. (ANOVA. Data represented as mean ± SEM. n = 6/C3a dose *p < 0.05, **p < 0.01). ( j ) mRNA expression of COL4 and COL6 normalized to GAPDH after treatment with C3a for 2 weeks in the absence and presence (pattern bar) of C3aR antagonist (ANOVA. n = 3/−C3aR ant and n = 3/+ C3aR ant. Data represented as mean ± SD, **p < 0.01, ***p < 0.001, ****p < 0.0001).

    Techniques Used: Immunolabeling, Expressing

    C3a does not make changes in calcium mobilization within the RPE cytosol. ( a ) Confocal fluorescent images of hfRPE cultures treated with different doses of rhC3a only (−C3aR ant) or rhC3a plus C3aR antagonist (+C3aR ant) after incubation with Fluoforte dye®. Scale bars 50 µm. ( b ) Quantification of calcium influx in hfRPE cultures treated with different doses of rhC3a (black bars) or rhC3a plus C3aR antagonist (grey bars), using fluorescent microscope or ( c ) microplate reader. (ANOVA, n = 8/treatment. Data represented as mean ± SD).
    Figure Legend Snippet: C3a does not make changes in calcium mobilization within the RPE cytosol. ( a ) Confocal fluorescent images of hfRPE cultures treated with different doses of rhC3a only (−C3aR ant) or rhC3a plus C3aR antagonist (+C3aR ant) after incubation with Fluoforte dye®. Scale bars 50 µm. ( b ) Quantification of calcium influx in hfRPE cultures treated with different doses of rhC3a (black bars) or rhC3a plus C3aR antagonist (grey bars), using fluorescent microscope or ( c ) microplate reader. (ANOVA, n = 8/treatment. Data represented as mean ± SD).

    Techniques Used: Incubation, Microscopy

    C3a decreases proteasome activity in the RPE, which is restored by the addition of C3aR antagonist. ( a ) Confocal images of UPP overall activity in hfRPE cells stimulated with different doses of C3a with or without C3aR antagonist for 72 hours stained with the probe MV-151. Control epox. = control samples treated overnight with epoxomicin. Scale bars 25 µm. Quantification of UPP overall activity after stimulation with different doses of C3a for ( b ) 2 weeks or ( c ) 72 hours with the probes MV-151 and LWA300, respectively. ( d ) mRNA expression of UPP subunits of hfRPE cells treated for 24 h with different doses of C3a normalized to GAPDH . (2-way ANOVA, n = 3/dose. Data represented as mean ± SD. *p < 0.05, **p < 0.01). White bars: control (C3a 0 ng/ml), grey bars (C3a 50 ng/ml), black bars (C3a 100 ng/ml).
    Figure Legend Snippet: C3a decreases proteasome activity in the RPE, which is restored by the addition of C3aR antagonist. ( a ) Confocal images of UPP overall activity in hfRPE cells stimulated with different doses of C3a with or without C3aR antagonist for 72 hours stained with the probe MV-151. Control epox. = control samples treated overnight with epoxomicin. Scale bars 25 µm. Quantification of UPP overall activity after stimulation with different doses of C3a for ( b ) 2 weeks or ( c ) 72 hours with the probes MV-151 and LWA300, respectively. ( d ) mRNA expression of UPP subunits of hfRPE cells treated for 24 h with different doses of C3a normalized to GAPDH . (2-way ANOVA, n = 3/dose. Data represented as mean ± SD. *p < 0.05, **p < 0.01). White bars: control (C3a 0 ng/ml), grey bars (C3a 50 ng/ml), black bars (C3a 100 ng/ml).

    Techniques Used: Activity Assay, Staining, Expressing

    C3a treatment results in increased MMP-2 activity by RPE cells. ( a ) mRNA expression of MMP-2 and TIMP-3 measured by qRT-PCR and normalized to GAPDH in cultures of hfRPE cells treated with different doses of C3a for two weeks. ( b ) Levels of TIMP-3 measured in conditioned media of the same cultures by ELISA. ( c ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with different doses of C3a for 2 weeks. ( d ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with or without C3aR antagonist for 2 weeks (ANOVA, n = 9/treatment. Data presented as mean ± SD. *p < 0.05).
    Figure Legend Snippet: C3a treatment results in increased MMP-2 activity by RPE cells. ( a ) mRNA expression of MMP-2 and TIMP-3 measured by qRT-PCR and normalized to GAPDH in cultures of hfRPE cells treated with different doses of C3a for two weeks. ( b ) Levels of TIMP-3 measured in conditioned media of the same cultures by ELISA. ( c ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with different doses of C3a for 2 weeks. ( d ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with or without C3aR antagonist for 2 weeks (ANOVA, n = 9/treatment. Data presented as mean ± SD. *p < 0.05).

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

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    Hycult Biotech c3ar, human, mab 17
    C3ar, Human, Mab 17, 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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    Hycult Biotech c3ar
    C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by <t>C3aR</t> antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.
    C3ar, 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
    https://www.bioz.com/result/c3ar/product/Hycult Biotech
    Average 90 stars, based on 1 article reviews
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    C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by C3aR antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3a causes abnormal deposition of ECM by hfRPE cells, which is prevented by C3aR antagonist. TEM images show transverse sections of hfRPE cultured on transwells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. ( d ), ( e ), and ( f ) are higher magnifications from white squares in ( a ), ( b ), and ( c ) respectively. C3a addition results in the accumulation of collagen fibers (white arrowheads) and wide-spaced collagen (black arrowheads) underneath the RPE cells. The addition of C3aR antagonist prevents the accumulation of sub-RPE deposits caused by C3a ( g – i ). Scale bars a–c: 2 µm, d–i: 500 nm.

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques: Cell Culture

    C3aR antagonist prevents formation of basal deposits caused by C3a. SEM images of decellularized transwells show exposed basal deposits made by hfRPE cells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. C3a triggers the accumulation of deposits made of ECM fibers underneath the RPE. The abnormal deposition is prevented by the addition of C3aR. Scale bars a–c: 100 µm, d–i: 10 µm.

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3aR antagonist prevents formation of basal deposits caused by C3a. SEM images of decellularized transwells show exposed basal deposits made by hfRPE cells treated for 2 weeks with 0 ( a , d , g ), 50 ( b , e , h ) or 100 ( c , f , i ) ng/ml of C3a in the absence ( a – f ) or presence ( g – i ) of 10 µM of C3aR antagonist. C3a triggers the accumulation of deposits made of ECM fibers underneath the RPE. The abnormal deposition is prevented by the addition of C3aR. Scale bars a–c: 100 µm, d–i: 10 µm.

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques:

    C3a induces overexpression of C3aR in the basolateral membrane of the RPE. ( a ) Immunolabeling with C3aR antibodies of hfRPE cells treated with different doses of C3a (C3aR-ant) or C3a + C3aR antagonist (C3aR + ant) for 2 weeks. Z-stack was built from images taken every 0.5 µm with confocal microscope. 90° projections show that C3aR is expressed on the basal-lateral membrane of RPE. Scale bars 50 µm. ( b ) Average quantification of C3aR fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a + C3aR antagonist (C3aR + ant). (ANOVA, n = 4/C3aR-ant and n = 4/C3aR + ant treatment. Data represented as mean ± SEM. *p < 0.05, **p < 0.01). ( c ) mRNA levels of C3aR of hfRPE cells treated with different doses of C3a for 24 hours (ANOVA, n = 6. Data presented as mean ± SD. **p < 0.01).

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3a induces overexpression of C3aR in the basolateral membrane of the RPE. ( a ) Immunolabeling with C3aR antibodies of hfRPE cells treated with different doses of C3a (C3aR-ant) or C3a + C3aR antagonist (C3aR + ant) for 2 weeks. Z-stack was built from images taken every 0.5 µm with confocal microscope. 90° projections show that C3aR is expressed on the basal-lateral membrane of RPE. Scale bars 50 µm. ( b ) Average quantification of C3aR fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a + C3aR antagonist (C3aR + ant). (ANOVA, n = 4/C3aR-ant and n = 4/C3aR + ant treatment. Data represented as mean ± SEM. *p < 0.05, **p < 0.01). ( c ) mRNA levels of C3aR of hfRPE cells treated with different doses of C3a for 24 hours (ANOVA, n = 6. Data presented as mean ± SD. **p < 0.01).

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques: Over Expression, Immunolabeling, Microscopy

    C3a causes specific deposition of Col IV and Col VI underneath the RPE that is prevented by C3aR antagonist. Immunolabeling with antibodies for ( a ) Col IV, ( c ) Col VI, ( e ) Col I, and ( g ) EFEMP1 of hfRPE cells treated with different doses of C3a or C3a + C3aR antagonist for 2 weeks. Images were taken with confocal. Orthogonal views show the basal deposition of the ECM proteins (left: apical, right: basal). Scale bars 50 µm. Average quantification of ( b ) Col IV, ( d ) Col VI, ( f ) Col I, and ( h ) EFEMP1 fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a plus C3aR antagonist (C3aR + ant) for 2 weeks. ( i ) Average quantification of immunostainings for ECM proteins after 4 weeks of treatment with C3a. (ANOVA. Data represented as mean ± SEM. n = 6/C3a dose *p < 0.05, **p < 0.01). ( j ) mRNA expression of COL4 and COL6 normalized to GAPDH after treatment with C3a for 2 weeks in the absence and presence (pattern bar) of C3aR antagonist (ANOVA. n = 3/−C3aR ant and n = 3/+ C3aR ant. Data represented as mean ± SD, **p < 0.01, ***p < 0.001, ****p < 0.0001).

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3a causes specific deposition of Col IV and Col VI underneath the RPE that is prevented by C3aR antagonist. Immunolabeling with antibodies for ( a ) Col IV, ( c ) Col VI, ( e ) Col I, and ( g ) EFEMP1 of hfRPE cells treated with different doses of C3a or C3a + C3aR antagonist for 2 weeks. Images were taken with confocal. Orthogonal views show the basal deposition of the ECM proteins (left: apical, right: basal). Scale bars 50 µm. Average quantification of ( b ) Col IV, ( d ) Col VI, ( f ) Col I, and ( h ) EFEMP1 fluorescent signal of hfRPE cultures treated with different doses of rhC3a (C3aR-ant) or rhC3a plus C3aR antagonist (C3aR + ant) for 2 weeks. ( i ) Average quantification of immunostainings for ECM proteins after 4 weeks of treatment with C3a. (ANOVA. Data represented as mean ± SEM. n = 6/C3a dose *p < 0.05, **p < 0.01). ( j ) mRNA expression of COL4 and COL6 normalized to GAPDH after treatment with C3a for 2 weeks in the absence and presence (pattern bar) of C3aR antagonist (ANOVA. n = 3/−C3aR ant and n = 3/+ C3aR ant. Data represented as mean ± SD, **p < 0.01, ***p < 0.001, ****p < 0.0001).

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques: Immunolabeling, Expressing

    C3a does not make changes in calcium mobilization within the RPE cytosol. ( a ) Confocal fluorescent images of hfRPE cultures treated with different doses of rhC3a only (−C3aR ant) or rhC3a plus C3aR antagonist (+C3aR ant) after incubation with Fluoforte dye®. Scale bars 50 µm. ( b ) Quantification of calcium influx in hfRPE cultures treated with different doses of rhC3a (black bars) or rhC3a plus C3aR antagonist (grey bars), using fluorescent microscope or ( c ) microplate reader. (ANOVA, n = 8/treatment. Data represented as mean ± SD).

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3a does not make changes in calcium mobilization within the RPE cytosol. ( a ) Confocal fluorescent images of hfRPE cultures treated with different doses of rhC3a only (−C3aR ant) or rhC3a plus C3aR antagonist (+C3aR ant) after incubation with Fluoforte dye®. Scale bars 50 µm. ( b ) Quantification of calcium influx in hfRPE cultures treated with different doses of rhC3a (black bars) or rhC3a plus C3aR antagonist (grey bars), using fluorescent microscope or ( c ) microplate reader. (ANOVA, n = 8/treatment. Data represented as mean ± SD).

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques: Incubation, Microscopy

    C3a decreases proteasome activity in the RPE, which is restored by the addition of C3aR antagonist. ( a ) Confocal images of UPP overall activity in hfRPE cells stimulated with different doses of C3a with or without C3aR antagonist for 72 hours stained with the probe MV-151. Control epox. = control samples treated overnight with epoxomicin. Scale bars 25 µm. Quantification of UPP overall activity after stimulation with different doses of C3a for ( b ) 2 weeks or ( c ) 72 hours with the probes MV-151 and LWA300, respectively. ( d ) mRNA expression of UPP subunits of hfRPE cells treated for 24 h with different doses of C3a normalized to GAPDH . (2-way ANOVA, n = 3/dose. Data represented as mean ± SD. *p < 0.05, **p < 0.01). White bars: control (C3a 0 ng/ml), grey bars (C3a 50 ng/ml), black bars (C3a 100 ng/ml).

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3a decreases proteasome activity in the RPE, which is restored by the addition of C3aR antagonist. ( a ) Confocal images of UPP overall activity in hfRPE cells stimulated with different doses of C3a with or without C3aR antagonist for 72 hours stained with the probe MV-151. Control epox. = control samples treated overnight with epoxomicin. Scale bars 25 µm. Quantification of UPP overall activity after stimulation with different doses of C3a for ( b ) 2 weeks or ( c ) 72 hours with the probes MV-151 and LWA300, respectively. ( d ) mRNA expression of UPP subunits of hfRPE cells treated for 24 h with different doses of C3a normalized to GAPDH . (2-way ANOVA, n = 3/dose. Data represented as mean ± SD. *p < 0.05, **p < 0.01). White bars: control (C3a 0 ng/ml), grey bars (C3a 50 ng/ml), black bars (C3a 100 ng/ml).

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques: Activity Assay, Staining, Expressing

    C3a treatment results in increased MMP-2 activity by RPE cells. ( a ) mRNA expression of MMP-2 and TIMP-3 measured by qRT-PCR and normalized to GAPDH in cultures of hfRPE cells treated with different doses of C3a for two weeks. ( b ) Levels of TIMP-3 measured in conditioned media of the same cultures by ELISA. ( c ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with different doses of C3a for 2 weeks. ( d ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with or without C3aR antagonist for 2 weeks (ANOVA, n = 9/treatment. Data presented as mean ± SD. *p < 0.05).

    Journal: Scientific Reports

    Article Title: C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway

    doi: 10.1038/s41598-018-28143-0

    Figure Lengend Snippet: C3a treatment results in increased MMP-2 activity by RPE cells. ( a ) mRNA expression of MMP-2 and TIMP-3 measured by qRT-PCR and normalized to GAPDH in cultures of hfRPE cells treated with different doses of C3a for two weeks. ( b ) Levels of TIMP-3 measured in conditioned media of the same cultures by ELISA. ( c ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with different doses of C3a for 2 weeks. ( d ) MMP-2 activity measured in apical and basal conditioned media of hfRPE cells treated with or without C3aR antagonist for 2 weeks (ANOVA, n = 9/treatment. Data presented as mean ± SD. *p < 0.05).

    Article Snippet: Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA).

    Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay