Journal: Scientific Reports

Article Title: Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

doi: 10.1038/srep30964

Figure Lengend Snippet: ( A , B ) 0.5 μg of human IgA1 was subjected to overnight digestion by 0.05 μg of IgA proteases from indicated bacterial strains. The digestion pattern was resolved by SDS-PAGE electrophoresis. The percentage of the residual heavy chain was indicated up the lane. A commercial IgA protease derived from N. gonorrhoeae was used as positive control. H stands for the heavy chain of IgA1 and L the light chain. Fc and Fd are the final products of digested IgA1 heavy chain. ( C) Quantification of catalytic activities of different bacterial IgA protease by ELISA-based assay. Data represent 4 independent replicates and each bar represents mean ± SEM for the percentage of reduction of OD value compared with the negative control (H 2 O). * P < 0.05 vs positive control. **P < 0.01 vs positive control. ***P < 0.001 vs positive control. ( D ) Dosage-dependent digestion of IgA1 by IgA proteases. 0.5 μg of IgA1 was incubated with different amount of IgA protease at 37 °C for 2 hours. The digestion mixture was subjected to SDS-PAGE analysis. The percentage of cleaved heavy chain (in relative to the negative control) was ploted. Note the ploted lines for commercial IgA protease, and proteases from N. gonorrhoeae 49226 , N. meningitidis 13090 , H. influenzae 49247 and 10211 were highlighted colorfully.

Article Snippet: H. influenzae with unkonwn serum type (ATCC49247), H. influenzae serum type b (ATCC 10211), S. pneumoniae serum type 3 (ATCC 6303TM), N. meningitidis serum type B (ATCC 13090TM) were bought from Chuanxiang Bio-tech Ltd (Shanghai, China).

Techniques: SDS Page, Electrophoresis, Derivative Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control, Incubation

Journal: Scientific Reports

Article Title: Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

doi: 10.1038/srep30964

Figure Lengend Snippet: ( A) Determination of glycosylation level of IgA1 after treatment with different deglycosylation enzyme (β-galactosidase and neuraminidase) by HHA-based ELISA. Each sample was assayed in triplicate and data was presented as mean ± SEM. *** P < 0.001 versus IgA1 group. ( B ) Western blots for determination of glycosylation level of modified IgA1 using biotin-labeled HAA to recognize GalNAc-exposed IgA1 (upper panel) and IgA1 specific antibody to detect total IgA1 (lower panel). Number below the lanes indicated the relative abundance of Galactose-deficient IgA1. The full-length blots were presented in . ( C) SDS-PAGE analysis of neuraminidase, β-galactosidase and IgA1 without treatment with the IgA protease. ( D ) SDS-PAGE analysis of IgA1 pre-treated with different deglycosylation enzymes and digested with IgA proteases from H. influenzae 10211 and 49247, N. meningitidis 13090 and N. gonorrhoeae 49226. PBS was used as a control. DesIgA1, desialylated IgA1. DeGalIgA1, degalactosylated IgA1. ( E )Quantitative analysis of the residual IgA1 (heavy chain) in Panel D . Data represent for 3 independent experiments. * P < 0.05 vs IgA1 group.

Article Snippet: H. influenzae with unkonwn serum type (ATCC49247), H. influenzae serum type b (ATCC 10211), S. pneumoniae serum type 3 (ATCC 6303TM), N. meningitidis serum type B (ATCC 13090TM) were bought from Chuanxiang Bio-tech Ltd (Shanghai, China).

Techniques: Glycoproteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Modification, Labeling, SDS Page, Control

Journal: Scientific Reports

Article Title: Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

doi: 10.1038/srep30964

Figure Lengend Snippet: agIgAs of different conformation were resolved in denature ( A ) and nature ( B ) PAGE gel. agIgAs of variable conformation were treated with IgA proteases from H. influenzae 10211 ( C ) and 49247 ( D ), N. meningitidis 13090 ( E ) and N. gonorrhoeae 49226 ( F ) followed by SDS-PAGE analysis. Total agIgA1 stands for total serum agIgA1and IC for agIgA1-IgG immune complexs. ( G ) Quantitative analysis of the residual IgA1 (heavy chain) in ( C–F ). Data represent for 3 independent experiments. * P < 0.05 vs total agIgA1 group, *** P < 0.001 vs total agIgA1 group.

Article Snippet: H. influenzae with unkonwn serum type (ATCC49247), H. influenzae serum type b (ATCC 10211), S. pneumoniae serum type 3 (ATCC 6303TM), N. meningitidis serum type B (ATCC 13090TM) were bought from Chuanxiang Bio-tech Ltd (Shanghai, China).

Techniques: SDS Page