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

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exogenous pulmonary surfactant prevents the development of intra‐abdominal adhesions in rats

doi: 10.1111/jcmm.12758

Figure Lengend Snippet: Expression and activity of MMP ‐9 in vivo . Gel zymography analysis showed that MMP ‐9 activity was significantly higher in GE ‐S versus GE animals ( P < 0.05) ( A ). In situ zymography analysis of MMP ‐9 expression showed a larger number of cells positive for gelatinase activity in GE ‐S versus GE animals. ( B ) Isolectin B4 ( IB 4) staining of the gastroenterostomy region revealed no significant difference in total number of macrophages ( C , arrows in the left‐hand column) between the GE and GE ‐S animals ( C , first‐ versus second‐row, and D ). The number of ED 1‐positive cells ( E , arrows in the left‐hand column) was significantly higher ( P < 0.001) in GE ‐S versus GE animals ( F ). No significant differences in number of cells double‐labelled for MMP ‐9/ IB 4 were observed between the GE ‐S and GE groups (third column in C ). Note that virtually all cells positive for ED 1 were MMP ‐9+ (third column in E ), scale bar: 200 μm.

Article Snippet: The expression and activity of MMP‐9 were investigated in all rats of groups L, LS, GE and GE‐S by SDS‐PAGE gelatin zymography using a 4% polyacrylamide stacking gel and a 10% polyacrylamide resolving gel (Bio‐Rad Co., Richmond, VA, USA) containing 1.5 mg/ml gelatin (BDH Laboratory Supplies, Poole, UK).

Techniques: Expressing, Activity Assay, In Vivo, Zymography, In Situ, Staining

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer

doi: 10.3390/ijms21124205

Figure Lengend Snippet: Schematic domain structure ( A ), gelatin zymography ( B ), and real-time gelatin zymography ( C ) of recombinant proMMP-9 variants. ( A ) At the top, the full-length proMMP-9 with its domains. Shown above in ( A ) is the numbering of the amino acids starting with the pre domain, which is cleaved off in the endoplasmatic reticulum, and the mature proenzyme, which starts at amino acid 20 and ends at amino acid 707. Shown also are the amino acids at the border of the FnII module and at the boarder of the OG and the HPX domain based on Vandooren et al. 2013 . The five deletion variants with their C-terminal amino acid are shown. In the fibronectin-deleted variants, the amino acids that were linked together after the FnII deletion are shown. The different recombinant proMMP-9 (rpMMP-9) variants were produced in Sf9 and High Five insect cells with a baculoviral transfection system as described in the Materials and Methods. ( B ) The purified variants were applied to gelatin zymography, and the rpMMP-9ΔFnII variant was from the pooled fractions from a Sepharose S-200 column. Under these conditions, this variant contained mainly the monomeric variant. ( C ) Real-time gelatin zymography of crude media of the three ΔFnII-deleted variants. Standards in ( B , C ) are: std-1 (a mixture of MMP-9 (homodimer/homotrimer 225 kDa; monomeric pro 92 kDa; and active 83 kDa) from THP-1 cells and MMP-2 (pro 72 kDa; active 62 kDa) from skin fibroblasts; std-2 (trypsin 24 kDa); and std-3 (catalytic domain of MMP-9 containing the FnII repeats 37 kDa). A line under gels indicates that the samples are from the same gel, which was cut and pasted for clarity in photoshop.

Article Snippet: Gelatin-Sepharose, heparin-Sepharose, Q-Sepharose, Sephadex G-50 (fine), Sephadex G-200, Sephacryl S-200, Sephacryl S-400, and protein G Sepharose were from GE-Healthcare (Uppsala, Sweden).

Techniques: Zymography, Recombinant, Produced, Transfection, Purification, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer

doi: 10.3390/ijms21124205

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of recombinant proMMP-9 variants. After purification, the different recombinant proMMP-9 (rpMMP-9) variants shown in were analyzed by Imperial stained SDS-PAGE ( A , B ) and Western blotting ( C ). To determine the purity, the molecular mass, as well as the presence of monomers and homomultimers, reducing (R) and non-reducing (NR) conditions were used. As a control, purified pMMP-9 (proMMP-9 from THP-1 cells) was used, which also contained small amounts of TIMP-1 (30 kDa), as seen in ( A ). Std-1 and Std-2 are the high range and broad range molecular weight standards from Thermo Scientific, respectively, with the molecular weights in kDa shown ( A , B ), and Std-3 in ( C ) is the biotinylated protein ladder. In ( C ), MMP-9ab and MMP-9HPXAb are polyclonal antibodies against the entire enzyme and the C-terminal HPX domain, respectively. In ( B , C ), recombinant proMMP-9ΔFnII is purified on either a Sephacryl-S-200 column (rpMMP-9ΔFnII (S-200)) or a heparin-Sepharose column (rpMMP-9ΔFnII (HS)), as described in the Materials and Methods. In ( C ), the crude media of two FnII-deletion variants (rpMMP-9ΔFnIIHPX and rpMMP-9ΔFnIIOGHPX) are used. In ( B ), the arrowhead shows the position of the rpMMP-9ΔFnII monomer. P1 and P2 are pooled non-reduced fractions from the S-200 column, and both contained rpMMP-9ΔFnII based on gelatin zymography. The line under gels indicates that the samples are from the same gel, which was cut and pasted for the sake of clarity in Photoshop.

Article Snippet: Gelatin-Sepharose, heparin-Sepharose, Q-Sepharose, Sephadex G-50 (fine), Sephadex G-200, Sephacryl S-200, Sephacryl S-400, and protein G Sepharose were from GE-Healthcare (Uppsala, Sweden).

Techniques: SDS Page, Western Blot, Recombinant, Purification, Staining, Molecular Weight, Zymography

Journal: PLoS ONE

Article Title: Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration

doi: 10.1371/journal.pone.0200872

Figure Lengend Snippet: A: Representative gelatin zymography analysis of LF fibroblasts in response to IL-6/sIL-6Rα stimulation for 24 h with or without Stattic. B: Quantitative analysis of data shown in panel A (n = 3). C: Concentration of soluble elastin following incubation for 6 h with the supernatant of IL-6-stimulated cultures with or without Stattic (n = 3). Data represent the mean ± SEM. **P < 0.01.

Article Snippet: LF fibroblasts with or without 50 nM Stattic pretreatment were stimulated with IL-6 (300 ng/ml) for 24 h. MMP-2 and -9 activities in the culture supernatant were measured with an SDS-PAGE gelatin zymography kit (Cosmo Bio, Tokyo, Japan) according to the manufacturer’s instructions.

Techniques: Zymography, Concentration Assay, Incubation