Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: Cleavage of purified C5 by thrombin, as detected by protein staining and Western blot. (A) SDS-PAGE on purified C5 (500 nM) from Comptech, Quidel, and our MgCl2 affinity-purified C5 eluted under mild conditions using a 2 M MgCl2 solution, incubated with thrombin (100 nM) for 0, 15, and 60 min at 37°C before the addition of lepirudin (1 μM). At 0 min, lepirudin was added before thrombin. Intact and cleaved α-chain is indicated by α and α′ respectively, β-chain is indicated with β. The gel was stained with Coomassie brilliant blue. (B) Purified C5 (500 nM) from Comptech was incubated with thrombin (100 nM) for different time intervals (7.5 min to 20 h), as indicated on top of a representative SDS-PAGE stained with Coomassie brilliant blue. Reference proteins are shown in separate lanes; thrombin (Thr), lepirudin (Lep), and C5 (C5), and an m.w. reference (M). C5 α-chain (α) and β-chain (β) are indicated in the label on the right-hand side of the figure, so are the primary (α′) and secondary (α′′ cleavage fragments, together with thrombin and C5a. (C) Purified Comptech C5 (500 nM) was incubated for 1 h at 37°C with PBS, thrombin (100 nM), thrombin (100 nM) in the presence of lepirudin (1 μM), or cobra venom factor (CVF). C5a-containing fragments were detected by Western blot using a polyclonal antibody against C5a. (D) GPRP-plasma was incubated with thrombin (400 nM) alone or in the presence of lepirudin (7 μM), purified Comptech C5 (60 μg/ml) alone or in the presence of thrombin, or the combination of purified C5 (60 μg/ml), thrombin (400 nM), and lepirudin (7 μM), for 16 h at 37°C. C5a-containing fragments were immunoprecipitated using a monoclonal antibody against C5a (clone 137-26) and detected by Western blot as described for (B). (E) Identical conditions as described for (C), but incubations were performed in serum from a C5-deficient individual instead of in GPRP-plasma. All samples were run under reducing conditions (A–E). M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Purification, Staining, Western Blot, SDS Page, Affinity Purification, Incubation, Combined Bisulfite Restriction Analysis Assay, Immunoprecipitation

Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: C5a Ag exposure and C5 cleavage in hydrochloric acid–acidified GPRP-plasma and serum. (A) Conformationally selective ELISA for detecting a C5a neoepitope of C5 in GPRP-plasma and C5-deficient serum including reconstitution with purified C5 (60 μg/ml) from Comptech. The plasma pH was adjusted with hydrochloric acid to 6.4, 6.8, or kept at 7.4, and incubated for 15 min at 37°C. Exposure of the C5a neoepitope in C5 was detected by ELISA, combining capturing and detecting Abs specific for the C5a neoepitope and C5b, respectively. Data are shown as the optical density mean values ± SD (n = 4), *p < 0.001. (B) GPRP-plasma was pH-adjusted to 6.4 and 6.8 with hydrochloric acid or kept at 7.4, and incubated with 0.9% NaCl, lepirudin (Lep) and/or thrombin (Thr, 400 nM) for 1 h at 37°C. C5a-containing fragments were specifically enriched by immunoprecipitation using a mAb against C5a (clone 137-26) and detected by Western blot, under reduced conditions, using a polyclonal Ab against C5a. Intact and cleaved C5 α-chain are indicated by α and α′, respectively, on the representative Western blot from two replicates. M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: C5a Ag exposure and C5 cleavage in hydrochloric acid– and lactic acid–acidified GPRP-plasma. (A and B) Conformationally selective ELISA for the detection of C5a neoepitope of C5 in normal GPRP-plasma (pH 7.4) and plasma acidified to pH 6.8 with hydrochloric acid (HCl) (A) or lactic acid (B), with or without neutralization to pH 7.4 with NaOH 1 min after acidification. Exposure of the C5a neoepitope in C5 was detected by ELISA, combining capturing and detecting Abs specific for the C5a neoepitope and C5b, respectively. Purified C5 (Comptech) at 60 μg/ml was included as a control. Data are shown as the optical density mean values ± SD (n = 3), *p < 0.001. (C) GPRP-plasma with and without lepirudin was pH-adjusted to 6.8 with hydrochloric acid (HCl) or lactic acid, with or without neutralization to pH 7.4 with NaOH 1 min after acidification. All samples, including GPRP-plasma, were kept at 7.4, and purified C5 with thrombin (400 nM) +/− lepirudin were incubated for 1 h at 37°C. C5a-containing fragments were specifically enriched by immunoprecipitation using a mAb against C5a (clone 137-26) and detected by Western blot, under reduced conditions, using a polyclonal Ab against C5a. Samples, as indicated on top of the membrane, are GPRP-plasma pH 7.4 (lanes 1 and 2), GPRP-plasma acidified to pH 6.8 with either hydrochloric acid (HCl) (lanes 3–6) or with lactic acid (lanes 7–10). Samples in lanes 4, 6, 8, and 10 are neutralized to pH 7.4 with NaOH after acidification. It is indicated which samples contain lepirudin. Intact and cleaved C5 α-chain are indicated by α and α′, respectively, on the representative Western blot from three replicates. M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Purification, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity

doi: 10.4049/jimmunol.2001471

Figure Lengend Snippet: Cleavage of C5 in clotting blood and thrombin-mediated cleavage of C5b in the C5b6 complex. (A) Human whole blood was collected in additive-free glass serum tubes. The blood was immediately acidified with 5% (v/v) lactic acid (0.165, 0.330, 0.500 M) or hydrochloric acid (HCl) (0.165, 0.330, 0.500 M) or added physiologic NaCl for volume control. The blood was let to clot for 60 min at 37°C and then centrifuged to serum. C5a-containing fragments were specifically enriched by immunoprecipitation using a mAb against C5a (clone 137-26) and detected by Western blot, under reduced conditions, using a polyclonal Ab against C5a. Intact and cleaved C5 α-chain are indicated by α and α′, respectively, on the representative Western blot from two replicates. (B) SDS-PAGE on purified C5b6 (50 μg/ml) from Comptech incubated with and without thrombin (400 nM) in PBS at pH 7.4, 6.8, and 6.4 for 60 min at 37°C. The samples were run on an SDS-PAGE under reduced conditions and stained with SYPRO Ruby Protein Gel Stain. Intact and cleaved α-chain is indicated by α and α′, respectively, β-chain is indicated with β, and C6 with C6. M, m.w. markers with size indicated in kDa.

Article Snippet: Proteins were transferred onto an immunoblot polyvinylidene fluoride membrane (Bio-Rad) and blotted using rabbit polyclonal IgG anti-prothrombin (Molecular innovations; Novi, MI) followed by an HRP-linked goat anti-rabbit-IgG (Southern Biotech, Birmingham, AL).

Techniques: Coagulation, Immunoprecipitation, Western Blot, SDS Page, Purification, Incubation, Staining

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Spearman correlations and population distribution of hepatic CYP2C8 phenotypes. CYP2C8 mRNA was determined by a specific TaqMan real-time RT-PCR assay, protein was determined by Western blotting and enzyme activity was measured by LC–MSMS analysis of amodiaquine N -desethylation in human liver microsomes ( n = 150). Results are means of duplicate measurements. (A–C) Spearman’s rank correlation coefficients are indicated as r s and statistical significance for all comparisons was p < 0.0001. (D) Histograms showing population distributions for mRNA (top), protein (middle), and enzyme activity (bottom) using 20 bins over the entire phenotype range.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Influence of non-genetic factors on CYP2C8 phenotype.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Influence of PPARα expression and genotype on the CYP2C8 phenotype. (A) Scatter plots of CYP2C8 phenotypes (amodiaquine N-desethylation, CYP2C8 protein and mRNA expression) vs. PPARα mRNA (top) and protein (bottom) expression. Spearman’s rank correlation coefficient r s and corresponding p -values are given. (B) Box-and-whisker plots of CYP2C8 phenotypes (Amodiaquine N -desethylation, CYP2C8 protein and mRNA expression) for two previously described intronic PPARA variants. Genotypes are indicated as G/G, G/A, and A/A (for rs4253728) and A/A, A/G, and G/G (for rs4823613). P (rec.), unadjusted p -value of Wilcoxon–Mann–Whitney test for recessive genetic model; N, number of individuals per group; numbers for the three genotype groups do not add up to 150 due to missing values. Outliers were included in all calculations.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Expressing, Whisker Assay, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: PPARα knockdown and activation in HepaRG cells. (A) mRNA levels were measured in HepaRG cultures cultured in three independent differentiation batches 72 h after PPARα activation using ligand WY14,643 (left part of the diagram) or siRNA-mediated PPARα knock-down (right part of the diagram) and compared with mRNA levels measured in cells treated with either DMSO (left) or non-targeting siRNA (right) set at 1.0. The graph shows the means from three (mRNA, activity) or two (protein) independent experiments, with error bars indicating standard deviations (mRNA, activity) and dots representing individual data points (protein). ∗ , Statistically significant ( P < 0.05, paired t -test). (B) Representative Western Blot analysis of the corresponding protein levels of CYP2C8 following either ligand-mediated induction (lanes DMSO and WY14,643) or siRNA-mediated knock-down (lanes siPPARa and siCTR) of PPARα.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Knockdown, Activation Assay, Cell Culture, Activity Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Binding of PPARα to the CYP2C8 promoter in vivo . Precipitated DNA from HepaRG cells without (w/o) treatment or after treatment with amodiaquine (10 μM for 6 h) was purified and was used, together with input DNA, as template for Sybr-Green PCR using a total of 12 primer pairs spanning approximately 10 kb of the CYP2C8 promoter region. Raw Ct (cycle threshold) values were normalized to input DNA to calculate the percentage of DNA immunoprecipitated. Primers encompassing the PPRE of the human HMGCR gene were used as positive control. Means relative to negative control primer pair (n.c.) are shown. (A–F) Schematic representation of CYP2C8 promoter regions, which were subjected for the ChIP analysis. Promoter scheme includes binding sites of previously described transcription factors.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Binding Assay, In Vivo, Purification, SYBR Green Assay, Immunoprecipitation, Positive Control, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Direct PPARα-mediated regulation of CYP2C8. (A) Electrophoretic mobility shift assays using in vitro translated proteins was used for assessment the binding of fluorescence-labeled double-stranded oligonucleotide probes corresponding to the indicated regions within CYP2C8 promoter (selection out of 15 tested probes is shown). As a positive control for binding, the known PPARα/RXRα binding site of the rat ACOX1 gene was used . Complexes of PPARα/RXRα heterodimers with the oligonucleotides are marked by an arrow. (B) Luciferase reporter gene constructs containing the sequences of the CYP2C8 promoter, PBR-A (–9500/–8500 bp) and PBR-C (–2500 to –3500 bp) were cotransfected with a PPARα expression plasmid and renilla-luciferase expression vector into HuH7 cells. Cells were treated with either 100 μM WY14,643 or vehicle DMSO for 48 h before measurement of firefly/renilla luciferase activities. Firefly luciferase activities were normalized to renilla luciferase activities to consider changes in the transfection variability and to DMSO control, set as 1. Data are means of three independent experiments, each performed in triplicates in 96-well format; (mut), mutated sites; ∗ , statistically significant ( P < 0.05) compared with DMSO treatment set to 1.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Electrophoretic Mobility Shift Assay, In Vitro, Binding Assay, Fluorescence, Labeling, Selection, Positive Control, Luciferase, Construct, Expressing, Plasmid Preparation, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Inhibitory role of β-catenin in the ligand-mediated induction of CYP2C8 by PPARα. (A) mRNA (gray bars) and protein (black bars) expression levels of CYP2C8 were analyzed by quantitative real-time qRT-PCR and Western blotting, respectively, following treatment of HepaRG cells with 100 μM of WY14,643 in the presence of β-catenin-targeting siRNA (siCatenin) or non-targeting control (siCTR). Shown are mean values of three independent experiments ±SD. ∗ , Statistically significant ( P < 0.05) compared with DMSO treatment set to 1; #, statistically significant ( P < 0.05) when siCatenin-treated cells compared to control siCTR-treated cells. (B) Representative western blot analysis of CYP2C8 protein expression following WY14,643-mediated activation of PPARα in the absence (lane siCatenin + WY14,643) or presence (lane siCTR + WY14,643) of β-catenin.

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

doi: 10.3389/fphar.2015.00261

Figure Lengend Snippet: Population variability of hepatic CYP2C8 expression phenotypes ( n = 150).

Article Snippet: Anti-human CYP2C8 monoclonal antibody (Rabbit anti-human CYP2C8, Puracyp # Hu-A004) and IRD800-labeled secondary anti-rabbit antibody (Li-cor) were used for detection with an Odyssey system (Li-cor).

Techniques: Expressing, Activity Assay