human c5 protein  (CompTech Computer Technologies)

Journal: The Journal of Biological Chemistry

Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils

doi: 10.1016/j.jbc.2024.107940

Figure Lengend Snippet: Norbin is required for the optimal agonist-induced internali z ation of C5aR1. A , schematic of the agonist-induced receptor internalization assay. B , bone marrow cells from Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) mice were incubated for 30 min at 37 °C. During this incubation, they were stimulated with 15 nM C5a for the indicated periods of time to induce the agonist-induced internalization of C5aR1. Cells were stained on ice to identify live neutrophils (Mac1 hi , Ly6G hi , FVD lo ) and quantify the level of C5aR1 on the neutrophil surface by flow cytometry. (i) The mfi of C5aR1 on the neutrophil surface was analyzed using FlowJo. (ii) To account for the increased basal cell surface level of C5aR1, data were normalized to 0 time. (iii) AUC of normalized cell surface C5aR1 over 30 min (iv) The mfi of Mac1 on the neutrophil surface was quantified as a control. (v) Level of C5aR1 on the neutrophil surface after incubation for 30 min at 37 °C, with either 15 nM or 100 nM C5a for the final 10 min of the incubation, as indicated. Data are mean ± SEM of five independent experiments (4 for 100 nM C5a, lower right); each dot is the mean of one experiment. Statistics are two-way ANOVA with Šidák’s multiple comparisons test, except (iii) which was two-tailed paired Student’s t test. p -values in black show significant differences, p -values in gr a y are not significant. C , cell fractionation. Purified Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) neutrophils were incubated for 30 min at 37 °C. Some were stimulated with 50 nM C5a for 1 or 10 min towards the end of the 30 min incubation to induce the agonist-induced internalization of C5aR1. Cells were lysed in detergent-free buffer and fractionated by differential centrifugation. The post-granule supernatant (PGS) was ultracentrifuged to generate cytosol (c) and post-granule membrane (m) fractions which were analyzed by SDS-PAGE and western blotting with antibodies against C5aR1, Gapdh as cytosol marker, and Kras as plasma membrane marker. 20% of the plasma membrane and 1.4% of the cytosol fractions were loaded. The abundance of C5aR1 in the plasma membrane fraction was quantified by Fiji densitometry and adjusted for the Coomassie signal over the whole lane to correct for protein loading (see also Fig. 7 ). Representative blots are shown. C5aR1 in the membrane fraction is expressed as % of the 0-time control. Data are mean ± SEM of six independent experiments. Statistics are two-way ANOVA with Šidák’s multiple comparisons test.

Article Snippet: Cells were incubated for 30 min at 37 °C and were then stimulated with the indicated concentrations of C5a (R&D systems, 2037-C5-025) or SDF1α (Peprotech, 250–20A) for the indicated periods of time, or were mock-stimulated.

Techniques: Incubation, Staining, Flow Cytometry, Control, Two Tailed Test, Cell Fractionation, Purification, Centrifugation, Membrane, SDS Page, Western Blot, Marker

Journal: The Journal of Biological Chemistry

Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils

doi: 10.1016/j.jbc.2024.107940

Figure Lengend Snippet: Norbin limits the C5a-stimulated recruitment of Tiam1, Vav1 and PKCδ to the plasma membrane, and suppresses the constitutive activity of Vav. A , (i) Cytosol (c) and post-granule membrane (m) fractions of C5a- or mock-stimulated Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) neutrophils from the same experiments as shown in Fig. 3 C were analyzed by SDS-PAGE and western blotting with antibodies against Tiam1, Vav, active phospho-Y173-Vav, and PKCδ. 20% of the membrane and 1.4% of the cytosol fractions were loaded. Blots showing the cytosol marker Gapdh and plasma membrane marker Kras are included for reference. Membranes were Coomassie-stained to control for protein loading. (ii) Blots were quantified by Fiji densitometry. Tiam1 and Vav in cytosol and membrane fraction were quantified as % of the PGS. phospho-Y173-Vav and PKCδ in the membrane fraction were normalized to the Coomassie signal over the whole lane. Data are mean ± SEM of 3 to 5 independent experiments, as indicated; each dot is the mean of one experiment. Statistics are two-way ANOVA with Šidák’s multiple comparisons test. p -values in black show significant differences, p -values in gr a y are not significant. B , norbin is largely cytosolic. Cytosol and post-granule membrane fractions from Ncdn fl/fl neutrophils were western blotted for Norbin. 20% of the plasma membrane and 1.4% of the cytosol fractions were loaded. C , norbin translocates from the plasma membrane into early endosomes upon C5a stimulation. Ncdn fl/fl neutrophils were stimulated for with 100 nM C5a for 2 min (n = 2) or 10 min (n = 1), or were mock-stimulated, and early endosomes were isolated from the PGS using Eea1 immunoprecipitation before the plasma membrane was purified from the endosome-depleted PGS by ultracentrifugation. The fractions were western blotted for Norbin, Kras and Eea1 as indicated. The same cell-equivalents of plasma membrane and endosome fractions were loaded. Representative blots shown are from 1 of 3 independent experiments. Norbin blots were quantified by Fiji densitometry. Date are mean and ± SEM of 3 independent experiments. Statistics are two-way ANOVA with Šidák’s multiple comparisons test.

Article Snippet: Cells were incubated for 30 min at 37 °C and were then stimulated with the indicated concentrations of C5a (R&D systems, 2037-C5-025) or SDF1α (Peprotech, 250–20A) for the indicated periods of time, or were mock-stimulated.

Techniques: Membrane, Activity Assay, SDS Page, Western Blot, Marker, Staining, Control, Isolation, Immunoprecipitation, Purification

Journal: The Journal of Biological Chemistry

Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils

doi: 10.1016/j.jbc.2024.107940

Figure Lengend Snippet: Norbin limits the recycling of internali z ed C5aR1 and CXCR4 back to the cell surface. A , schematic of the receptor recycling assay. B and C , bone marrow cells from Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) mice were stimulated for 10 min at 37 °C with high doses of agonist ( B : 100 nM C5a; C : 100 ng/ml nM SDF1α) to induce the maximal internalization of C5aR1 and CXCR4, respectively, or were mock-stimulated. Cells were washed, and then either kept on ice as a control, or incubated at 37 °C for the indicated periods of time to allow the recycling of internalized receptors back to the cell surface and were then kept on ice. Cells were stained on ice to identify live neutrophils (Mac1 hi , Ly6G hi , FVD lo ) and quantify the level of ( B ) C5aR1 or ( C ) CXCR4 on the neutrophil surface by flow cytometry. (i) The mfi of C5aR1 and CXCR4 on the neutrophil surface was analyzed using FlowJo. (ii) To account for the increased basal cell surface level of the GPCRs, data were normalized by setting the maximally internalized level to 0 and basal receptor levels to 100%. (iii) AUC of normalized cell surface C5aR1 and CXCR4 over 60 min (iv) The mfi of Mac1 on the neutrophil surface was quantified as a control. Data are mean ± SEM of 4 independent experiments for each receptor; each dot is the mean of one experiment. Statistics are two-way ANOVA with Šidák’s multiple comparisons test, except middle right, two-tailed paired Student’s t test. p -values in black show significant differences, p -values in gr a y are not significant.

Article Snippet: Cells were incubated for 30 min at 37 °C and were then stimulated with the indicated concentrations of C5a (R&D systems, 2037-C5-025) or SDF1α (Peprotech, 250–20A) for the indicated periods of time, or were mock-stimulated.

Techniques: Control, Incubation, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils

doi: 10.1016/j.jbc.2024.107940

Figure Lengend Snippet: Norbin is required for sustained β-arrestin recruitment and promotes the agonist-induced internali z ation of C5aR1 in a β-arrestin-dependent manner. A , β-arrestin recruitment. Purified Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) neutrophils were incubated for 10 min at 37 °C while being stimulated with 15 nM C5a for 0, 0.17, 1 or 10 min, as indicated. Cells were lysed, a post-granule supernatant (PGS) was prepared by centrifugation, and glycosylated proteins, including C5aR1, were purified from the PGS using wheat-germ agglutinin agarose. Proteins were analyzed by SDS-PAGE and western blotting with C5aR1 and β-arrestin (β-Arr) antibodies. Representative western blots are shown. Blots were quantified by Fiji densitometry, and the β-arrestin signal was normalized to the amount of C5aR1 in each lane. Data are mean ± SEM of five independent experiments; each dot is the mean of one experiment. Statistics are two-way ANOVA with Šidák’s multiple comparisons test. p -values in black show significant differences, p -values in gr a y are not significant. B , β-arrestin-dependent agonist-induced internalization. Bone marrow cells were pretreated with 100 μM barbadin for 30 min at 37 °C ( filled symbols ), or mock-treated with 1% DMSO ( open symbols ), and stimulated for 5 min with 50 nM C5a, or mock-stimulated (control). Cells were stained on ice to identify live neutrophils (Mac1 hi , Ly6G hi , FVD lo ) and quantify the level of C5aR1 on the neutrophil surface by flow cytometry. The mfi of C5aR1 and Mac1 on the neutrophil surface were analyzed using FlowJo. Data are mean ± SEM of 3 independent experiments; each dot is the mean of one experiment. Statistics are two-way ANOVA with Šidák’s multiple comparisons test. p -values in black show significant differences, p -values in gr a y are not significant. C , barbadin blocks the recruitment of the clathrin-adaptor AP2. Purified Ncdn fl/fl neutrophils were incubated for 30 min at 37 °C in the presence of 100 μM barbadin or mock-treated, prior to stimulation with 15 nM C5a for 1 min at 37 °C. Glycosylated proteins purified from the PGS using wheat-germ agglutinin agarose as in ( A ) and analyzed by western blotting with AP2α1 antibody. Coomassie staining was used as a loading control. The blot shown is representative of two independent experiments. D , receptor degradation. Purified neutrophils were incubated for 2 h at 37 °C while being stimulated with 15 nM C5a for the indicated periods of time towards the end. Cells were fixed, permeabilized, and stained to analyse the total cellular level of C5aR1 by flow cytometry. Left-hand panel : The mfi of total neutrophil C5aR1 was analyzed using FlowJo. Right-hand panel : AUC of the 2 h time course. Data are mean ± SEM of three independent experiments; each dot is the mean of one experiment. Statistics are two-tailed paired Student’s t test. p -values in gr a y are not significant.

Article Snippet: Cells were incubated for 30 min at 37 °C and were then stimulated with the indicated concentrations of C5a (R&D systems, 2037-C5-025) or SDF1α (Peprotech, 250–20A) for the indicated periods of time, or were mock-stimulated.

Techniques: Purification, Incubation, Centrifugation, SDS Page, Western Blot, Control, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils

doi: 10.1016/j.jbc.2024.107940

Figure Lengend Snippet: Norbin suppresses Erk and p38 Mapk signal ing and Gα i -dependent ROS production in C5a-stimulated neutrophils. A , Norbin suppresses Erk and p38 Mapk signaling. Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) neutrophils were stimulated with 15 nM C5a as indicated, lysed, and analyzed by western blotting for phosphorylated, active and total Erk, p38 Mapk and Akt. Representative blots are shown. Quantification by ImageJ densitometry of phospho-signals divided by total protein. Data are mean ± SEM of three independent experiments. Statistics are two-way ANOVA with Šidák’s multiple comparisons test. p -values in black show significant differences, p -values in gr a y are not significant. B , norbin suppresses Gα i -dependent ROS production. ROS production in the presence of pertussis toxin (PTX) was measured in purified Ncdn fl/fl ( black ) and Ncdn Δmye ( red ) neutrophils using real-time chemiluminescence assay. Neutrophils were pre-incubated with increasing concentrations of PTX, as indicated, primed with TNFα/GM-CSF, and stimulated with 25 nM C5a. ( left ) ROS production was quantified by integrating the area under the curve (AUC) of the ROS response over 5 min and plotted as a function of the PTX concentration. Date are mean and ± SEM of three independent experiments. Statistics are two-way ANOVA with Šidák’s multiple comparisons test. p -values showing significant differences are indicated. ( right ) Data were normalized to the mock-treated condition for each genotype, and best-fit curves ( dotted lines ) and IC 50 s were determined using GraphPad.

Article Snippet: Cells were incubated for 30 min at 37 °C and were then stimulated with the indicated concentrations of C5a (R&D systems, 2037-C5-025) or SDF1α (Peprotech, 250–20A) for the indicated periods of time, or were mock-stimulated.

Techniques: Western Blot, Purification, Chemiluminescence Immunoassay, Incubation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Anaphylatoxins C3a and C5a differentially regulate endothelial monoculture dnCI in a dose-dependent manner. ( A , B ) Monitoring of dnCI changes in response to 50 nM, 100 nM and 500 nM of anaphylatoxins C3a and C5a revealed a transient decrease in dnCI by 500 nM C5a after 2 h of treatment in HBMEC. At 24 h after anaphylatoxin stimulation, 500 nM C3a significantly decreased the paracellular resistance in HBMEC compared to the untreated control. ( C , D ) In HREC, we observed no significant alterations in paracellular resistance after 2 h of C3a or C5a treatment. A dose-dependent increase in dnCI occurred 24 h after treatment initiation in HREC treated with 50 nM C3a, 100 nM C3a, 50 nM C5a and 100 nM C5a compared to the untreated control. ( A , C ) ↑ indicates analyses time points at 2 and 24 h. All data were normalized to the respective cell index (CI) value before the addition of treatment and to the untreated control (dnCI). ( A , B ) RTCA graph represents data from n = 10 for untreated and 500 nM C3a, n = 6 for 50 nM C3a and 50, 100 and 500 nM C5a and n = 8 for 100 nM C3a treated cells, respectively. ( C , D ) RTCA graph represents data from n = 9 for untreated, n = 6 for 50 nM C5a and 50, 100 and 500 nM C3a, n = 8 for 100 nM C5a and n = 4 for 500 nM C5a, respectively. ( B , D ) Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test was used for datasets including non-parametric data. One-way analysis of variance (ANOVA) with Dunnett’s T3 post hoc test was used for parametric data sets. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C3a and C5a do not change CDH5 gene expression but alter VE-cadherin on protein level in HBMEC and HREC. ( A , B ) Treatment with 500 nM C3a or 500 nM C5a did not change CDH5 gene expression in HBMEC and HREC compared with the untreated control. ( C ) Two hours after treatment, HBMEC exhibited a heterogeneous VE-cadherin phenotype in untreated and 500 nM C3a-treated cells. HBMEC treated with 500 nM C5a displayed a more homogeneous distribution of VE-cadherin. VE-cadherin mean fluorescent intensity (mean FI) remained stable regardless of treatment. Two hours after treatment, HREC displayed a homogeneous monolayer in all treatment groups without disruption of the endothelial monolayer. VE-cadherin signal intensity was increased by 500 nM C5a. ( D ) After 24 h, HBMEC displayed a homogeneous phenotype in untreated and 500 nM C5a-treated cells, but they were elongated and showed disrupted VE-cadherin expression in 500 nM C3a-treated cells (white arrowheads). VE-cadherin signal intensity was decreased by 500 nM C3a but was increased by 500 nM C5a. In HREC, the homogenous phenotype remained stable over 24 h. 500 nM C3a increased VE-cadherin signal intensity. ( A , B ) Data were curated from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. ( C , D ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Control, Disruption

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Anaphylatoxin treatment caused increased C3 expression in HREC and increased C3AR1 expression in HBMEC. ( A , B ) qRT-PCR analysis revealed no effect of anaphylatoxin treatment on C3 expression in HBMEC but disclosed an increase in C3 expression in 500 nM C5a-treated HREC 2 h post-treatment and a 500 nM C3a and 500 nM C5a mediated increase in C3 gene expression 24 h post-treatment in HREC. ( C , D ) Two hours’ exposure to 500 nM C5a increased C3AR1 expression in HBMEC, while anaphylatoxin treatment had no significant effect on C3AR1 expression in HREC. ( A – D ) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test for datasets including non-parametric data. One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C3a presence increased in HREC after exposure to C3a and C5a. ( A ) After 2 h, the C3 protein signal intensity increased in C3a- and C5a-treated HBMEC. The C3a signal in untreated HBMEC exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution. In HREC, C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups after 2 h, but the signal intensity increased in C3a- and C5a-treated HREC. HBMEC and HREC C3a signal intensity remained stable across treatment groups and exhibited a spot-wise pattern across all treatment groups after 2 h. ( B ) After 24 h, the C3 signal decreased in C3a-treated HBMEC and returned to baseline in C5a-treated cells. In untreated HBMEC, C3a exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution 24 h post-treatment initiation. The localization of C3a transitioned from spot-wise distribution to a more diffuse cellular distribution in HBMEC treated with 500 nM C3a and 500 nM C5a 24 h after treatment initiation. HREC C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups. C3 signal intensity increased in C3a- and C5a-treated HREC after 24 h. The spot-wise C3a distribution pattern seen after 2 h persisted in untreated HREC 24 h after treatment but expanded to a broader, cell-covering signal in cells treated with 500 nM C3a and 500 nMC5a. C3a signal intensity increased in C3a- and C5a-treated HREC 24 h after treatment initiation. ( A , B ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C5 gene and protein expression are elevated in HREC following C5a stimulation, whereas no significant changes are observed in HBMEC. ( A , B ) qRT-PCR indicated no variations in C5 gene expression among treatment groups or analysis time points at 2 and 24 h in HBMEC. Two hours post-treatment, exposure to 500 nM C3a and 500 nM C5a resulted in a significant upregulation in C5 gene expression in HREC. This effect was reversed 24 h post-treatment. ( C , D ) Stimulation with 500 nM C3a and 500 nM C5a provoked no change in C5 protein signal strength in HBMEC. Two hours post-treatment, C5 exhibited a spot-wise expression pattern in both untreated and 500 nM C3a-treated HREC, whereas HREC treated with 500 nM C5a showed a broader distribution of immunofluorescent signals. C5a increased C5 signal intensity compared to the untreated control and C3a-treated HREC. After 24 h, C5 detection signal returned to baseline in C3a- and C5a-treated cells. ( A , B ) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. ( C , D ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C5AR1 transcript expression remains unchanged, but protein detection changes under anaphylatoxic stress. ( A , B ) qRT-PCR analysis showed no difference in C5AR1 gene expression between untreated and C3a- or C5a-treated HBMEC and HREC. ( C ) Treatment with 500 nM C3a and 500 nM C5a reduced C5aR1 protein signal in HBMEC after 2 h of treatment. Immunofluorescent staining of C5aR1 in HREC showed a stable and comparable signal after 2 h in all treatment groups. ( D ) After 24 h, the C5aR1 signal was stable between treatment groups in HBMEC. C5aR1 protein signal intensity increased in 500 nM C5a-treated HREC after 24 h. ( A , B ) Data were curated from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. ( C , D ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Adding 50 nM of C5a, but not 100 nM or 500 nM, counteracted the barrier-disruptive effect of 500 nM C3a and improved the HBMEC barrier after 24 h. ( A , B ) Monitoring of combined anaphylatoxin-treated dnCI changes in HBMEC revealed a C5a dependent increase in dnCI, which counteracted the barrier-disruptive effect of C3a. 24 h post-anaphylatoxin stimulation, HBMEC treated with 500 nM C3a + 50 nM C5a reached a significantly higher dnCI compared to 500 nM C3a-treated cells. ( C , D ) RTCA measurement revealed an increase in dnCI in HREC treated with 500 nM C3a + 100 nM C5a 2 h after treatment addition compared to 500 nM C3a-treated cells. This regulation was maintained for 24 h after exposure to treatment. ( E ) Immunocytochemistry revealed a homogeneous endothelial monolayer in all treatment groups in HBMEC, 2 h post-treatment. ( F ) Twenty-four hours post-treatment, 500 nM C3a decreased the VE-cadherin signal intensity, whereas treatment with 500 nM C3a + 50 nM C5a obtained a VE-cadherin signal strength similar to that of the untreated control. ( A , C ) ↑ indicate analyses time points 2 and 24 h. All data were normalized to the respective CI value before addition of treatment and to the untreated control. ( A , B ) RTCA graph represents the mean of data from n = 9 for 500 nM C3a, n = 6 for 500 nM C5a and n = 7 for 500 nM C3a + 50 nM C5a and 500 nM C3a + 100 nM C5a and n = 4 for 500 nM C3a + 500 nM C5a-treated cells, respectively. ( C , D ) RTCA graph represents the mean of data from n = 6 for 500 nM C3a, 500 nM C3a + 50 nM C5a and 500 nM C3a + 100 nM C5a and n = 4 for 500 nM C5a and n = 5 for 500 nM C3a + 500 nM C5a. ( B , D ) Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test for datasets including non-parametric data. One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. ( E , F ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Immunocytochemistry, Control

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: 500 nM C3a and C5a increase the transcellular permeability for IgG in HBMEC but not in HREC, reflecting reduced cell–cell contact integrity at 500 nM C3a but not enhanced paracellular resistance at 500 nM C5a in HBMEC. ( A ) Endothelial cells were allowed to settle and form a continuous monolayer 17 h prior to the addition of 500 nM C3a or 500 nM C5a. After 24 h, 100 µg/mL purified IgG was applied luminally, and abluminal supernatants were harvested 2 h later to assess IgG migration through the endothelial barrier using Western blot detection. ( B ) Western blot quantification revealed a significant increase in IgG migration through the endothelial barrier in 500 nM C3a- and 500 nM C5a-treated HBMEC compared to the untreated control. This effect was not observed in HREC, where the IgG migration rate was similar to the untreated control in cells treated with 500 nM C3a and 500 nM C5a. Bar height represents the mean relative protein level for n = 3 for all treatment groups in both cell types. Cell types were analyzed as separate datasets. Uncropped Western blots are shown in . One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Permeability, Purification, Migration, Western Blot, Control

Journal: bioRxiv

Article Title: Asparagine couples group A Streptococcal metabolism to virulence

doi: 10.1101/2024.07.08.602371

Figure Lengend Snippet: (a,b) Degradation of recombinant C5a by cultures supernatant containing the C5a-peptidase enzyme (ScpA). GAS was grown in CDM without or with Asn or/and LL-37. Supernatants of the indicated strains were incubated with recombinant human C5a, resolved on Tris-tricine gels, and visualized by Coomassie blue staining. M represents a marker, and an empty arrow is the cleaved C5a. The data are representative of 2 independent experiments. (c,d) Quantitation of IL-8 degradation by ScpC present in the supernatants of the indicated strains by ELISA. n=2 . The data shown represent the means ± S.D. (e) Asn does not affect in-vitro CovR phosphorylation . Purified CovR was incubated in the absence (−) and presence (+) of acetyl phosphate (Ac-P) as a phosphate donor and the indicated concentrations of Asn (µg ml -1 ). The protein samples were resolved on Phos-tag SDS-PAGE gel and visualized.

Article Snippet: The cell-free supernatants were incubated with 1 mg/ml of purified recombinant human complement component C5a (R&D Systems, USA) at 37°C for 2 h. The samples were heated at 100°C for 5 min with 4x Tricine loading buffer (0.4 M Tris HCl pH 6.8, 80% glycerol, 4% SDS, and 0.08% Coomassie blue) to stop the reaction.

Techniques: Recombinant, Incubation, Staining, Marker, Quantitation Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Purification, SDS Page