Journal: Frontiers in Immunology

Article Title: Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese

doi: 10.3389/fimmu.2022.973169

Figure Lengend Snippet: Associations of CFB gene polymorphisms with the risk of IgAN.

Article Snippet: A eukaryotic expression plasmid containing the coding sequence of CFB , either the rs12614-C allele (CS-Q0537-M35) or the rs12614-T allele (EX-Q0537-M35), was constructed (GeneCopoeia, MD, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese

doi: 10.3389/fimmu.2022.973169

Figure Lengend Snippet: Associations of SNP rs12614 with clinicopathological parameters in IgAN cases.

Article Snippet: A eukaryotic expression plasmid containing the coding sequence of CFB , either the rs12614-C allele (CS-Q0537-M35) or the rs12614-T allele (EX-Q0537-M35), was constructed (GeneCopoeia, MD, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese

doi: 10.3389/fimmu.2022.973169

Figure Lengend Snippet: Comparison of serum levels of CFB, CFH, and C3a among different genotypes of rs12614. The serum levels of CFB (A, B) , CFH (C, D) , and C3a (E, F) in patients with IgAN and healthy controls among different genotypes of rs12614 (CC or CT+TT). The bars represent the mean (standard deviation) or median (interquartile range). The P -value was determined by Student’s t -test or the non-parametric Mann-Whitney U -test.

Article Snippet: A eukaryotic expression plasmid containing the coding sequence of CFB , either the rs12614-C allele (CS-Q0537-M35) or the rs12614-T allele (EX-Q0537-M35), was constructed (GeneCopoeia, MD, USA).

Techniques: Comparison, Standard Deviation, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese

doi: 10.3389/fimmu.2022.973169

Figure Lengend Snippet: Effect of rs12614 (R32W mutation) on the stability of the CFB protein. (A) System flexibility analysis of CFB 32R and CFB 32W . A molecular docking search was performed followed by all-atom, explicit water MD simulations. The root mean square deviation (RMSD) of the backbone of CFB 32W (3.5107 Å) was larger than that of CFB 32R (2.7145 Å). (B, C) Final stable structures of CFB 32R (B) and CFB 32W (C) . A clustering strategy was applied to the MD trajectory. The cluster center after the equilibrium of the system was selected as the final stable complex of luteolin with CFB 32R and CFB 32W . The 10-residue-length loops containing the R32 and W32 are colored in purple.

Article Snippet: A eukaryotic expression plasmid containing the coding sequence of CFB , either the rs12614-C allele (CS-Q0537-M35) or the rs12614-T allele (EX-Q0537-M35), was constructed (GeneCopoeia, MD, USA).

Techniques: Mutagenesis, Residue

Journal: Frontiers in Immunology

Article Title: Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese

doi: 10.3389/fimmu.2022.973169

Figure Lengend Snippet: Effect of rs12614 on the expression of CFB protein. Plasmids carrying rs12614-C (CFB 32R ) or rs12614-T (CFB 32W ) were transfected into HEK293 cells. The expression levels of CFB in the cell supernatants were measured by ELISA. The data were replicated in three independent experiments. The bars represent median (interquartile range). The P -value was tested by the non-parametric Mann-Whitney U -test.

Article Snippet: A eukaryotic expression plasmid containing the coding sequence of CFB , either the rs12614-C allele (CS-Q0537-M35) or the rs12614-T allele (EX-Q0537-M35), was constructed (GeneCopoeia, MD, USA).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Malaysia/B HA is not activated by murine tPA, uPA, LTF, NSP4, CFB, tryptase ϵ, and HGFA. A, examination of HA cleavage by soluble proteases present in cell supernatants. HEK293 cells with transient expression of Malaysia/B HA were incubated with cleared protease containing HEK293 cell supernatants as described under “Experimental procedures” (left) or recombinant rKLK8 (right). Treatment of HA-expressing cells with buffer (w/o) or trypsin was used as control. Cell lysates were analyzed for HA cleavage by immunoblotting. β-Actin was used as loading control. B, MDCK cells with transient protease expression were infected with Malaysia/B at a low MOI of 0.01 and incubated for 24 h to allow multicycle viral replication. Cells transfected with empty vector (ev) or murine TMPRSS2-expressing plasmid were used as control. Virus spread was visualized by immunostaining of infected cells against NP. C, expression analysis of tryptase ϵ_DDDDK mutant in HEK293 cells with or without enterokinase treatment. Supernatants of cells transfected with empty vector (ev) or tryptase ϵ_DDDDK-encoding plasmid were concentrated (5×) at 48 h post-transfection and analyzed by SDS-PAGE and Western blotting using tryptase ϵ–specific antibodies. Zymogen and mature form are indicated by filled and open arrowheads, respectively. D, examination of HA cleavage by tryptase ϵ. HEK293 cells expressing Malaysia/B HA were incubated with tryptase ϵ_DDDDK mutant–containing cell supernatants treated with or without enterokinase (10 IU). Treatment of HA-expressing cells with trypsin was used as control. Cell lysates were analyzed for HA cleavage. E, expression analysis of HGFA in HEK293 supernatants with and without matriptase treatment. At 48 h post-transfection with empty vector (ev) or HGFA-encoding plasmid cell supernatants were concentrated (5×), treated with or without matriptase (5.0 μg/ml) for 1 h at 37 °C, and analyzed by immunoblotting using a FLAG-specific antibody. Zymogen and mature form are indicated by filled and open arrowheads, respectively. F, examination of HA cleavage by HGFA. HEK293 cells co-transfected with plasmids encoding Malaysia/B HA and either empty vector (w/o) or HGFA-encoding plasmid were incubated with exogenous matriptase or trypsin (0.5 μg/ml each) or remained untreated for 24 h. Cell lysates were analyzed for HA cleavage by Western blotting.

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Expressing, Incubation, Recombinant, Western Blot, Infection, Transfection, Plasmid Preparation, Immunostaining, Mutagenesis, SDS Page