rat c5ar  (Hycult Biotech)

Journal: In Vivo

Article Title: Renal Expression Levels of C5a Receptor and Autophagy‐related Beclin‐1 and LC3A/B Are Simultaneously Enhanced Under Immunoglobulin Treatment in a Rat Model of Sepsis

doi: 10.21873/invivo.13883

Figure Lengend Snippet: Kidney expression levels of C5a receptor (C5aR), LC3A/B and beclin-1 evaluated using immunoblotting, quantified by measuring band intensity, normalized by β-actin expression, and expressed as arbitrary units in control, sham, cecal ligation perforation (CLP)+saline, CLP+IgG and CLP+IgGAM treatment arms. Vertical bars indicate standard deviations. Symbols above the vertical bars denote significant differences between the IgG and IgGAM treatment groups vs. control (*), sham (#) and CLP+saline (†) groups. Significant differences between CLP+IgG and CLP+IgGAM groups are denoted by § at the 1 st (24 h) (1d, green) and 10 th (10d, blue) days of the experiment. *,#,†,§ p<0.05; **,##,††,§§ p<0.01; ***,###,††† p<0.001.

Article Snippet: Then, anti-rat C5aR (Thermo Fisher Scientific), Beclin-1 (Cell Signaling, Danvers, MA, USA) and LC3A/B (Cell Signaling) primary antibodies were applied in 2.5% skim milk and the membrane was incubated at +4 ̊C overnight. β-actin (Abcam) was used as the reference protein.

Techniques: Expressing, Western Blot, Control, Ligation, Saline

Journal: In Vivo

Article Title: Renal Expression Levels of C5a Receptor and Autophagy‐related Beclin‐1 and LC3A/B Are Simultaneously Enhanced Under Immunoglobulin Treatment in a Rat Model of Sepsis

doi: 10.21873/invivo.13883

Figure Lengend Snippet: Representative immunoblotting bands for kidney expression levels of C5a receptor (C5aR), LC3A/B and beclin-1 in each group and the 1 st day (1d) and 10 days (10d) in control, sham, cecal ligation perforation (CLP)+saline, CLP+IgG and CLP+IgGAM treatment arms. β-actin was used as the reference protein.

Article Snippet: Then, anti-rat C5aR (Thermo Fisher Scientific), Beclin-1 (Cell Signaling, Danvers, MA, USA) and LC3A/B (Cell Signaling) primary antibodies were applied in 2.5% skim milk and the membrane was incubated at +4 ̊C overnight. β-actin (Abcam) was used as the reference protein.

Techniques: Western Blot, Expressing, Control, Ligation, Saline

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: A schematic view of the experimental design. Cells were obtained from the hippocampal formation of rat embryos (E18.5) using enzymatic and mechanical dissociation methods. Neuron-astrocyte co-cultures were used to investigate C5aR expression/activation and the effect of C5a on cell viability. The microfluidic device was used to quantify the C5a effect on axonal growth.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Activation Assay

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: Primers used for gene expression by RT-PCR.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: C5aR expression in neuron-astrocyte co-cultures by RT-PCR. ( A ) C5aR mRNA expression was observed in both injured and intact cell cultures. Controls included expression of housekeeping GAPDH mRNA. ( B ) Double immunostaining for NF-L (green) and C5aR (red) indicated that injured and intact neurons expressed NF-L ( a , e ) and C5aR ( b , f ) on their membranes ( n = 3). Merged images in yellow ( c , g ) revealed a co-localization of these markers. Controls for each condition were performed by omitting the primary antibodies ( d , h ) and nuclei were counterstained using DAPI. The dotted lines and * indicate the site of injury. Scale bars: 50 µm. ( C ) Double immunostaining of NF-L in red ( a , e ) and C5aR in green ( b , f ) on spinal cord cryosections. Controls were performed by omitting the primary antibodies ( d , h ) and nuclei were counterstained in blue using DAPI. Images revealed that C5aR was co-expressed with NF-L in grey and white matters (yellow in ( c , g )). Scale bars: 50 µm.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Double Immunostaining

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: Interaction between C5a and neuron C5aR. Cells were cultured in the B-27-supplemented neurobasal medium as a control condition without C5a ( a ), stimulated with C5a (50 ng/mL) ( b ) or with both C5a and PMX53 ( c ) for 5 min ( n = 3). Immunostaining of the phosphorylated C5a receptor (C5aR-P, red) and NF-L (green) indicated that while NF-L was expressed in all conditions, C5aR-P was not expressed by cells when the inhibitor was added. Scale bars: 20 µm.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Cell Culture, Control, Immunostaining

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: Axonal growth in the microfluidic device. ( A ) Cells were incubated with the control medium for 48 h either without C5a ( a , d ), or with C5a (50 ng/mL) ( b , e ), or with both C5a and PMX53 ( c , f ). GFP labeling allowed visualization of the axonal growth via pictures taken at 40×. The axonal growth is reported by combining the images obtained over the 48 h growth period. Scale bars: 100 µm. ( B ) After axonal injury, the axonal length significantly increased under all conditions between 24 h (white) and 48 h (hatching). Adding C5a significantly increased the axonal length at 24 h and at 48 h as compared to the controls at the same delays. This increase was suppressed after adding the C5aR inhibitor. Results are presented as boxplots. The central line in each boxplot represents the median values while the box edges indicate the first and third quartiles. All points beyond the whiskers up to 1.5 times the interquartile range are considered as outliers. # indicates a significant ( p < 0.05) difference between incubation periods for the same treatment. * and + indicate significant ( p < 0.05) differences between the axonal lengths after adding C5a as compared to the control at 24 and 48 h, respectively. $ and & indicate significant ( p < 0.05) differences with C5a + PMX53 compared to the C5a treatment at 24 and 48 h, respectively. Ө indicates significant ( p < 0.05) differences with C5a + PMX53 compared to the control at 24 h.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Incubation, Control, Labeling

Journal: PLoS ONE

Article Title: Osteoblast-specific overexpression of complement receptor C5aR1 impairs fracture healing

doi: 10.1371/journal.pone.0179512

Figure Lengend Snippet: (A) Representative histological images of wildtype (WT) and Col1a1-C5aR1 mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E) Bone mineral density (BMD). (F) Relative flexural rigidity. (G) Number of osteoblasts per bone perimeter (N.Ob/B.Pm) and (H) number of osteoclasts per bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aR mice. (I) Representative tartrate-resistant acid phosphatase (TRAP) staining of fractured calli of WT and Col1a1-C5aR1 mice with isolated fracture. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale bar 100 μm, * p < 0.05; ** p < 0.005; *** p < 0.001, n = 6–7 per group.

Article Snippet: Primary rat anti-mouse C5aR (CD88) (MA1-81761, 1:1000, ThermoFisher Scientific), rabbit anti-mouse p-Erk1/2 (4370P, 1:1000, Cell Signaling Technology), rabbit anti-mouse Erk1/2 (4695P, 1:1000, Cell Signaling Technology), rabbit anti-mouse p-p38 (4511P, 1:1000, Cell Signaling Technology), rabbit anti-mouse p38 (8690P, 1:1000, Cell Signaling Technology) and a rabbit anti-mouse GAPDH antibody (2118, 1:2000, Cell Signaling) were incubated overnight at 4°C, followed by incubation with a secondary HRP-coupled anti-rat or anti-rabbit antibody (1:15,000, Cell Signaling Technology), respectively, at RT for 1 h. WesternBright TM Quantum or WesternBright TM ECL chemiluminescent HRP substrate (both Advansta, CA, USA) were added to the membranes for 2 min at RT and the luminescent signal was captured using the Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany) or by membrane exposure to X-ray film.

Techniques: Isolation, Staining