unconjugated mouse anti human complement c9 antibody  (Hycult Biotech)

Journal: PLoS ONE

Article Title: Aluminum Hydroxide Adjuvant Differentially Activates the Three Complement Pathways with Major Involvement of the Alternative Pathway

doi: 10.1371/journal.pone.0074445

Figure Lengend Snippet: The alternative pathway (AP) is activated when C3 undergoes spontaneous hydrolysis and forms the initial C3 convertase, C3(H 2 O)Bb in the presence of factor B (fB) and cleavage of bound fB by factor D (fD). The alternative C3-convertase is stabilized by properdin (P). The C3 convertase generates C3b and the subsequent C3-convertases are assembled by C3b and Bb. The lectin pathway (LP) is activated when MBL or other immune lectins bind carbohydrates on pathogens, activating the associated serine proteases (MASPs) which cleave C4 and C2. The first component of the classical pathway (CP) is C1, a complex of C1q and its associated serine proteases C1r and C1s. The CP is initiated by C1q recognition of immune complexes, activating C1r and C1s, again cleaving C4 and C2 generating a C3 convertase characteristic of the CP and the LP (C4bC2a). The C3 convertase cleaves C3 generating C3b and enables assembly of a C5 convertase (C3bC3bBb or C4bC2aC3b), and the C5-convertase product (C5b) initiates assembly of the membrane attack complex (MAC) from C5b-C9. * Initial alternative C3 convertase generated from C3(H 2 O) and fB.

Article Snippet: Mouse anti-human C5b-C9 (membrane attack complex (MAC)) and rabbit anti-human C3c were from Dako (Glostrup, Denmark).

Techniques: Generated

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: HBV diminishes complement C9 expression. Relative mRNA expression of different membrane attack complex components in a Huh7 cells transfected with full-length HBV linear monomer relative to untransfected cells (control) and b HepG2 hNTCP cells infected with cell culture-derived HBV particles in comparison to uninfected HepG2 hNTCP cells (control) as measured by Real-time PCR. c Serum concentration of C9 in IT, EP-CHB, IC, EN-CHB and HC detected by ELISA. d Relative mRNA expression of C9 in chronically HBV infected liver biopsy tissues compared to control liver tissues. e Relative mRNA expression of C9 in Huh7 cells transfected separately with plasmids encoding all three HBV surface proteins (HBs), HBV core (HBc), HBV Polymerase (HBp) or HBV X (HBx). f Expression of C9 protein in HBx or empty vector transfected Huh7 cells assayed by immunoblot with anti-C9 primary antibody; cellular α-Tubulin served as the loading control. g Relative mRNA expression of C9 when transfected with different concentrations (0, 400, 600 and 1000 ng/ml) of pcDNA3.1/myc-His(B)-HBx plasmid. mRNA expression was normalized with endogenous 18S ribosomal RNA value. In cases of a , b and d – g , values represent data from three independent experiments, mean ± SD. For c statistical significance was assessed by one-way ANOVA followed by Tukey's Multiple Comparison Test (** p < 0.005 and *** p < 0.0001). Paired t -tests were performed for comparisons of paired groups in a, b and d-f. ** p < 0.005, *** p < 0.0001

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Infection, Cell Culture, Derivative Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: HBX downregulates C9 promoter activity and USF-1 expression. a Relative expression of C9 in HBx transfected Huh7 cells with or without DNA methylation inhibitor 5-aza-2’-deoxycytidine (5 μM) or histone deacetylation inhibitor Trichostatin A (0.1 μM). b Relative luciferase activity of C9 promoter-luciferase reporter construct (pGL3-C9-Prom) when co-transfected with HBx-expressing plasmid [pcDNA3.1/myc-His(B)-HBx] or empty vector. c CpG island prediction in C9 promoter using Methprimer online software. Relative expression of d USF1and ATF2 mRNA and e USF-1 protein in HBx- or empty vector transfected Huh7 cells measured respectively by real time PCR and western blot analysis using anti-USF1 antibody. Mean ± SD are presented based on three independent experiments. Paired t tests were performed for comparing paired groups. * p < 0.05, *** p < 0.0001

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: Activity Assay, Expressing, Transfection, DNA Methylation Assay, Luciferase, Construct, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: USF-1 is an important transcription factor that regulates C9 expression and is inhibited by HBX. a Relative luciferase activity of C9 promoter in Huh7 cells transfected with pcDNA3.1/myc-His(B)-HBx, pGL3-C9-Prom and pRL-CMV Renilla Luciferase Reporter vector along with different concentrations (0, 400, 800 and 1200 ng/ml) of pCMV-USF1 plasmid. b Relative C9 mRNA expression following transfection of Huh7 cells with pcDNA3.1/myc-His(B)-HBx and pCMV-USF1 plasmid in different concentrations (0, 400, 800 and 1200 ng/ml). c Relative C9 promoter luciferase activity in Huh 7 cells transfected with pGL3-C9-Prom, pRL-CMV Renilla Luciferase Reporter vector and different concentrations of antisense oligonucleotide designed against USF-1 gene (USF-1_ASO) (0–1200 ng/ml). d Relative C9 mRNA expression following transfection of Huh7 cells with USF-1_ASO (1000 ng/ml) or scrambled oligo control (ASO_Ctrl) (1000 ng/ml). e Assessment of relative C9 luciferase activity upon transfection of Huh7 cells with wild-type pGL3-C9-Prom and its mutated derivative pGL3-C9-Prom-mt in presence or absence of USF-1 expressing plasmid (pCMV-USF1). f ChIP analysis of USF1 binding to C9 promoter in HBx and empty vector transfected Huh7 cells. ChIP analysis was performed using IgG (negative control) and anti-USF-1 antibody. The relative enrichment of C9 promoter DNA was normalized to the input DNA (5%) for each experiment. Mean ± SD are based on three independent experiments. Paired t tests were performed for comparing paired groups in d , f . Comparisons between groups were performed using the one-way ANOVA with p values adjusted by the Tukey’s multiple comparison test in e . ** p < 0.005, *** p < 0.0001

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Negative Control

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: HBX limits USF-1 availability for C9 transcription by hypermethylation of USF-1 promoter. a CpG island prediction in USF-1 promoter using Methprimer online software. b Relative expression of USF1 and C9 in HBx transfected Huh7 cells with or without DNA methylation inhibitor 5-aza-2′-deoxycytidine (5 μM). c Relative expression of DNMT3A in Huh7 cells transfected with HBX-expressing plasmid and empty vector. d Schematic representation of CpG dinucleotides at USF-1 promoter in Huh7 cells transfected with HBX-expressing plasmid and empty vector. The horizontal line represents the sequencing result of one clone, and the vertical line represents each individual CpG sites. The open circles represent unmethylated cytosine and the closed circles represent methylated cytosine. The percentages of methylated CpGs in USF-1 promoter in case of HBx and empty vector transfected Huh7 cells were calculated and compared. Values represented as mean ± SD. Paired t tests were performed for comparing paired groups. ** p < 0.005, *** p < 0.0001

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: Software, Expressing, Transfection, DNA Methylation Assay, Plasmid Preparation, Sequencing, Methylation

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: Reduced MAC formation and Huh7 cell lysis with CHB sera were reversed upon C9 supplementation. a Representative immunofluorescence images at ×40 magnification showing MAC formation (C5b-9, green) on HBV transfected Huh7 cells treated with serum from HC and CHB patient as well as CHB serum supplemented with C9 protein. The nucleus was counterstained with DAPI (blue). b Viability of HBV transfected Huh7 cells measured by MTT assay upon incubation with HC sera, CHB sera and C9 supplemented CHB sera. The absorbance readings were averages of triplicate experiments. c Correlation analysis between serum C9 concentration and HBV-DNA levels in chronically HBV infected individual. One-way ANOVA followed by Tukey’s multiple comparison were used in (b). ** p < 0.005, *** p < 0.0001

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: Lysis, Immunofluorescence, Transfection, MTT Assay, Incubation, Concentration Assay, Infection

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: Impairment of MAC mediated bacterial killing by CHB patient sera. a C9 deposition on E. coli treated with CHB patient sera in comparison to HC sera. b CFU count of E. coli on agar plates incubated at 37 °C for 16 h after treatment with HC sera, C9 deficient CHB sera or heat inactivated HC sera. Determination of c Bacterial-DNA by Real-time PCR and d endotoxin concentration by ELISA in the sera of IT, CHB (including both EP- and EN-CHB), IC and HC. Correlation analysis between serum C9 level and e bacterial DNA and f endotoxin titre in study subjects. Paired t test was performed in a . One-way ANOVA followed by Tukey’s multiple comparison were used in (b)-(d). * p < 0.05, ** p < 0.005, *** p < 0.0001

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: Incubation, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biomedical Science

Article Title: Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

doi: 10.1186/s12929-022-00876-1

Figure Lengend Snippet: Recovery of serum C9 level and reduction in viral/bacterial/endotoxin load in CHB patients after Tenofovir-therapy. Levels of a HBV-DNA, b alanine aminotransferase (ALT), c C9, d bacterial-DNA and e endotoxin in sera of CHB patients at baseline and after 12 months of Tenofovir therapy. Wilcoxon matched paired t tests were performed. * p < 0.05, ** p < 0.005

Article Snippet: Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology).

Techniques: