Journal: Journal of Clinical Immunology

Article Title: Testing the Activity of Complement Convertases in Serum/Plasma for Diagnosis of C4NeF-Mediated C3 Glomerulonephritis

doi: 10.1007/s10875-016-0290-5

Figure Lengend Snippet: Mechanism of convertase stabilization by C4NeF isolated from patient seven. a Spontaneous decay of classical C3 convertase assembled from purified components. Sensitized erythrocytes were coated with C1 and C4. Addition of C2 initiated the process of convertase formation. Convertase activity was assessed at indicated time points in the same manner, as described in Figs. and . b The same experiment as in a but with CD55-Fc added together with C2. c Representative blot of the C4b degradation pattern (C4b, factor I, and C4BP present) and comparison to negative control with no factor I added and sample, in which C4b cleavage was inhibited by excessive anti-C4c antibody. d Quantification of C4d produced upon C4b degradation in the presence of Igs isolated from patient seven or from NHS. When anti-C4c antibodies were tested in the same experiments, C4d readout was below the lower level of detection and therefore is not shown. All graphs show data collected from at least 3 independent experiments and statistical significance was assessed with two-way ANOVA assay at * p < 0.05, ** p < 0.0,1 and *** p < 0.001

Article Snippet: Human sera depleted from C3 and/or C5 as well as mouse anti- C4d antibody (A253) were purchased from Quidel.

Techniques: Isolation, Purification, Activity Assay, Comparison, Negative Control, Produced

Journal: Arthritis Research & Therapy

Article Title: The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

doi: 10.1186/s13075-016-1123-x

Figure Lengend Snippet: A scheme of the complement system. Complement can be activated via three different routes. The classical and lectin pathways have their own specific pattern recognition molecules (PRMs), whereas the alternative pathway is activated by a spontaneous tick-over of C3 and deposition of C3b molecules onto permissive surfaces, which is facilitated by properdin (P). All pathways converge at the stage of C3 convertases, which catalyse breakdown of C3 into C3a and C3b molecules. When C3b molecules bind to the convertase complex, it gives rise to the C5 convertase. Except for the triggers indicated for each pathway in the scheme, apoptotic and necrotic cellular debris as well as degradative protein fragments from the extracellular matrix can also trigger all three pathways [ , ]. In red and boldface type are the markers of complement activation analysed in our study: C4d, the end degradation product of C4b; C3bBbP, a soluble form of alternative C3 convertase including properdin; and sTCC, soluble terminal complement complex (a soluble form of C5b-9). Factor D Serine protease that cleaves factor B, Igs Immunoglobulins, MASP Mannose-binding lectin-associated serine protease, MBL Mannose-binding lectin

Article Snippet: Briefly, for the C4d assay, a capture antibody against a C4d neoepitope, together with a mouse anti-C4d detection antibody (A253; Quidel, San Diego, CA, USA), followed by peroxidase-conjugated goat anti-mouse (P0447; Dako, Carpinteria, CA, USA) were used.

Techniques: Activation Assay, Binding Assay

Journal: Arthritis Research & Therapy

Article Title: The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

doi: 10.1186/s13075-016-1123-x

Figure Lengend Snippet: Synovial fluid concentrations of C4d, C3bBbP and soluble terminal complement complex (sTCC). a Box plots with subjects ordered by the diagnostic groups: reference, osteoarthritis (OA), rheumatoid arthritis (RA), pyrophosphate arthritis (PPA), and knee injuries split into recent injury and old injury. b Knee injury samples ordered by days after injury in subgroups of 24–46 subjects (Table ), showing recent injury and old injury groups. Boxes show the quartiles (median, 25th and 75th percentiles) with error bars and whiskers for the 10th and 90th percentiles. The quartiles of the reference group are extended as thin horizontal lines in both panels for comparison. Statistically significant group differences, determined by Mann-Whitney U test, versus the reference group are indicated by asterisks (*) and are presented in Additional file : Table S1. CAU Complement activation units

Article Snippet: Briefly, for the C4d assay, a capture antibody against a C4d neoepitope, together with a mouse anti-C4d detection antibody (A253; Quidel, San Diego, CA, USA), followed by peroxidase-conjugated goat anti-mouse (P0447; Dako, Carpinteria, CA, USA) were used.

Techniques: Diagnostic Assay, Comparison, MANN-WHITNEY, Activation Assay

Journal: Arthritis Research & Therapy

Article Title: The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

doi: 10.1186/s13075-016-1123-x

Figure Lengend Snippet: Correlation between C4d, C3bBbP and sTCC in reference and patient groups

Article Snippet: Briefly, for the C4d assay, a capture antibody against a C4d neoepitope, together with a mouse anti-C4d detection antibody (A253; Quidel, San Diego, CA, USA), followed by peroxidase-conjugated goat anti-mouse (P0447; Dako, Carpinteria, CA, USA) were used.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

doi: 10.1186/s13075-016-1123-x

Figure Lengend Snippet: Correlation between C4d, C3bBbP, sTCC and other biomarkers in the recent injury group

Article Snippet: Briefly, for the C4d assay, a capture antibody against a C4d neoepitope, together with a mouse anti-C4d detection antibody (A253; Quidel, San Diego, CA, USA), followed by peroxidase-conjugated goat anti-mouse (P0447; Dako, Carpinteria, CA, USA) were used.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: The complement system is activated in synovial fluid from subjects with knee injury and from patients with osteoarthritis

doi: 10.1186/s13075-016-1123-x

Figure Lengend Snippet: C4d, C3bBbP, sTCC concentrations in the recent injury group in relation to structural features

Article Snippet: Briefly, for the C4d assay, a capture antibody against a C4d neoepitope, together with a mouse anti-C4d detection antibody (A253; Quidel, San Diego, CA, USA), followed by peroxidase-conjugated goat anti-mouse (P0447; Dako, Carpinteria, CA, USA) were used.

Techniques:

Journal: International Journal of Cancer

Article Title: Involvement of cyclin d1-cdk5 overexpression and mcm3 cleavage in bax-associated spontaneous apoptosis and differentiation in an a253 human head and neck carcinoma xenograft model

doi: 10.1002/(sici)1097-0215(19991029)83:3<341::aid-ijc9>3.0.co;2-3

Figure Lengend Snippet: FIGURE 3 – Western blot analysis to detect bcl-2/bax and p53/WAF1 protein expression. Xenografts were removed at indicated times after transplantation. Xenograft lysates were examined using anti-bcl-2, anti-bax, anti-p53 and anti-WAF1 antibodies, followed by incubation with secondary antibody conjugated to peroxidase as described in Material and Methods. The relative positions on the blot of protein m.w. markers are shown in kilodaltons on the right. b-Tubulin signals were also obtained to ensure equivalency of protein loading.

Article Snippet: Primary antibodies used for characterization of A253 xenografts in immunoblot analysis are as follows: mouse anti-human bcl-2 monoclonal antibody (MAb) (Dako, Carpinteria, CA) and polyclonal rabbit anti-human bax (a kind gift of Dr. J.C. Reed, La Jolla, CA and PharMingen, San Diego, CA); polyclonal rabbit antihuman bax (P-19, Santa Cruz Biotechnology, Santa Cruz, CA) for further characterization of the specificity of bax expression; mouse anti-human p53 MAb (DO1, Santa Cruz Biotechnology) and polyclonal rabbit anti-human p53 (FL393, Santa Cruz Biotechnology); mouse anti-human WAF1 MAb (Oncogene Research, Cambridge, MA), mouse anti-human cyclin D1 MAb (R124), polyclonal rabbit anti-cdk4 (C22) and -cdk5 (C8) (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Transplantation Assay, Incubation

Journal: International Journal of Cancer

Article Title: Involvement of cyclin d1-cdk5 overexpression and mcm3 cleavage in bax-associated spontaneous apoptosis and differentiation in an a253 human head and neck carcinoma xenograft model

doi: 10.1002/(sici)1097-0215(19991029)83:3<341::aid-ijc9>3.0.co;2-3

Figure Lengend Snippet: FIGURE 6 – Changes in cell cycle regulatory protein expression after transplantation.A253 xenografts were removed at the indicated time points after transplantation, followed by immunoblot analysis for cyclin D1, cyclin-dependent kinase-5 (cdk5) and cdk4 as described previously. Xenograft lysates were analyzed using polyclonal rabbit anti-human cdk5, polyclonal rabbit anti-human cdk4 or mouse anti-human cyclin D1 monoclonal antibodies as described in Material and Methods. b-Tubulin signals are shown for equal loading.

Article Snippet: Primary antibodies used for characterization of A253 xenografts in immunoblot analysis are as follows: mouse anti-human bcl-2 monoclonal antibody (MAb) (Dako, Carpinteria, CA) and polyclonal rabbit anti-human bax (a kind gift of Dr. J.C. Reed, La Jolla, CA and PharMingen, San Diego, CA); polyclonal rabbit antihuman bax (P-19, Santa Cruz Biotechnology, Santa Cruz, CA) for further characterization of the specificity of bax expression; mouse anti-human p53 MAb (DO1, Santa Cruz Biotechnology) and polyclonal rabbit anti-human p53 (FL393, Santa Cruz Biotechnology); mouse anti-human WAF1 MAb (Oncogene Research, Cambridge, MA), mouse anti-human cyclin D1 MAb (R124), polyclonal rabbit anti-cdk4 (C22) and -cdk5 (C8) (Santa Cruz Biotechnology).

Techniques: Expressing, Transplantation Assay, Western Blot, Bioprocessing

Journal: International Journal of Cancer

Article Title: Involvement of cyclin d1-cdk5 overexpression and mcm3 cleavage in bax-associated spontaneous apoptosis and differentiation in an a253 human head and neck carcinoma xenograft model

doi: 10.1002/(sici)1097-0215(19991029)83:3<341::aid-ijc9>3.0.co;2-3

Figure Lengend Snippet: FIGURE 7 – Cyclin D1 and cyclin-dependent kinase-5 (cdk5) activity analysis. A253 xenografts were removed at the indicated time points as described in Figure 6 and immunoprecipitation analysis was carried out as described in Material and Methods. Three hundred micrograms of xenograft extracts were precipitated with the indicated antibodies. Two micrograms of histone H1, as the substrate, were incubated with each immunoprecipitate plus 5 µCi [32P]-gATP for 20 min at 30°C. The reaction mixture was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the extent of histone H1 phosphorylation was detected by autoradiography.

Article Snippet: Primary antibodies used for characterization of A253 xenografts in immunoblot analysis are as follows: mouse anti-human bcl-2 monoclonal antibody (MAb) (Dako, Carpinteria, CA) and polyclonal rabbit anti-human bax (a kind gift of Dr. J.C. Reed, La Jolla, CA and PharMingen, San Diego, CA); polyclonal rabbit antihuman bax (P-19, Santa Cruz Biotechnology, Santa Cruz, CA) for further characterization of the specificity of bax expression; mouse anti-human p53 MAb (DO1, Santa Cruz Biotechnology) and polyclonal rabbit anti-human p53 (FL393, Santa Cruz Biotechnology); mouse anti-human WAF1 MAb (Oncogene Research, Cambridge, MA), mouse anti-human cyclin D1 MAb (R124), polyclonal rabbit anti-cdk4 (C22) and -cdk5 (C8) (Santa Cruz Biotechnology).

Techniques: Activity Assay, Immunoprecipitation, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Phospho-proteomics, Autoradiography

Journal: International Journal of Cancer

Article Title: Involvement of cyclin d1-cdk5 overexpression and mcm3 cleavage in bax-associated spontaneous apoptosis and differentiation in an a253 human head and neck carcinoma xenograft model

doi: 10.1002/(sici)1097-0215(19991029)83:3<341::aid-ijc9>3.0.co;2-3

Figure Lengend Snippet: FIGURE 8 – Correlation between bcl-2, bax, cyclin D1 and cyclin-dependent kinase-5 (cdk5) with ladder-type DNA fragmentation. Bcl-2, bax and cyclin D1-cdk5/b-tubulin ratio (——) and ladder-type DNA fragmentation and cytokeratin expression (-----) are plotted as a function of days after transplantation in A253 xenografts. The procedures for determining gene expression and DNA fragmentation are described in detail in Figures 1 and 2. Relative expression levels of gene products are determined by scanning the films shown in Figures 3 and 6. Semi-quantitation assay of ladder-type DNA fragmentation was carried out on signal intensity of 4 bands from the bottom of the gel (Fig. 1). Relative apoptotic DNA fragmentation are determined by the sum of the intensity of these 4 bands.

Article Snippet: Primary antibodies used for characterization of A253 xenografts in immunoblot analysis are as follows: mouse anti-human bcl-2 monoclonal antibody (MAb) (Dako, Carpinteria, CA) and polyclonal rabbit anti-human bax (a kind gift of Dr. J.C. Reed, La Jolla, CA and PharMingen, San Diego, CA); polyclonal rabbit antihuman bax (P-19, Santa Cruz Biotechnology, Santa Cruz, CA) for further characterization of the specificity of bax expression; mouse anti-human p53 MAb (DO1, Santa Cruz Biotechnology) and polyclonal rabbit anti-human p53 (FL393, Santa Cruz Biotechnology); mouse anti-human WAF1 MAb (Oncogene Research, Cambridge, MA), mouse anti-human cyclin D1 MAb (R124), polyclonal rabbit anti-cdk4 (C22) and -cdk5 (C8) (Santa Cruz Biotechnology).

Techniques: Expressing, Transplantation Assay, Gene Expression, Quantitation Assay