Anti-MMP3 Rabbit Monoclonal Antibody

Western blot analysis of MMP3 using anti-MMP3 antibody (M00775).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: rat PC-12 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MMP3 antigen affinity purified monoclonal antibody (M00775) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for MMP3 at approximately 50 kDa. The expected band size for MMP3 is at 50 kDa.

Immunofluorescent analysis of HeLa cells, using MMP3 Antibody.

Immunohistochemical analysis of paraffin-embedded human liver cancer, using MMP3 Antibody.

The role of TRADD in TNF-α-mediated cellular events. The cells were transfected with siRNA and lipofectamine 3000 for 48 h. After 48 h transfection, the medium was replaced by refresh medium and chondrocytes were treated with TNF-α (5 ng/ml) for 24 h, the protein expression of TRADD, iNOS, COX2, COL2A1, MMP13, cleaved-caspase-3, and LC3 was examined ( A – C , E , F ). After 48 h transfection with siRNA and lipofectamine 3000, the medium was replaced by refresh medium and chondrocytes were pre-treated with z-VAD for 2 h and then exposed to TNF-α for 24 h, the phosphorylation of RIPK1, RIPK3, and MLKL as well as protein expression of these three markers were detected using western blot ( G ). I , K The protein expression of iNOS, COX2, COL2A1, and MMP3 was examined in chondrocytes overexpressing TRADD after TNF-α intervention. D , H , J , L Semi-quantitative analysis of protein bands by image J and data in figures were expressed as means ± SD. * p < 0.05, ** p < 0.01. NS not significant,. NC negative control. z-VAD, Z-VAD-FMK. C-caspase-3, cleaved-caspase-3. M The graphical scheme summarized the findings of the role of TRADD in TNF-α-mediated chondrocytes events.
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W-Exo-L@GelMA exhibits a strong chondrocyte-targeting effect and a pronounced action on promoting anabolism and suppressing catabolism and inflammation without causing the inhibition of chondrocyte viability. A Cell viability assessed by CCK8 assay. No obvious cytotoxicity on chondrocytes was observed when treated with W-Exo-L@GelMA loaded with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 48 h. Data represent mean ± SD; N = 6/group; one-way ANOVA; ns, not significant. B Immunofluorescence of Dil-labeled exosomes. The uptake of exosomes was observed in the chondrocytes when treated with Exo-L, Exo-L@GelMA or W-Exo-L@GelMA for 48 h. Dil was used for labeling exosomes (red), DAPI to label nuclei (blue), and Phalloidin to label the cytoskeleton (green). Scar bar: 200 μm. C Western blot analyses of the protein levels of anabolic, catabolic, and inflammatory factors in the IL-1β-induced chondrocytes treated with W-Exo-L@GelMA loaded with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 48 h. W-Exo-L@GelMA promoted COL2 and SOX9 and inhibited iNOS, COX2, MMP3, and MMP13 protein levels in a dose-dependent manner. D Quantitative analysis of the western blot results. Data represent mean ± SD; N = 3/group; *P < 0.05; **P < 0.01 by one-way ANOVA
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LRRK2-IN-1 suppresses the IL-1β-induced inflammation and catabolism and induces anabolism without causing the inhibition of chondrocyte viability. A Schematic diagram of cell treatment and experimental procedures. B Cell viability assessed by CCK8 assay. No obvious inhibition of chondrocyte proliferation was observed when treated with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 24 h. Data represent mean ± SD; N = 6/group; one-way ANOVA; ns, not significant. C Western blot analyses of the protein levels of anabolic, catabolic, inflammatory factors in the IL-1β-induced chondrocytes treated with 0.5, 1.0, 2.5, and 5.0 µM LRRK2-IN-1 for 24 h. LRRK2-IN-1 suppressed MMP3, MMP13, iNOS, and COX2 and induced COL2 and SOX9 in a dose-dependent manner. D Quantitative analyses of the western blot results. Data represent mean ± SD; N = 3/group; *P < 0.05; **P < 0.01 by one-way ANOVA. E Immunofluorescence of iNOS, MMP13, and aggrecan expression in the IL-1β-induced chondrocytes treated with 5.0 µM LRRK2-IN-1 for 24 h. Scar bar: 400 μm
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LIPUS inhibits the progression of OA. A , B Western blot and quantitative analysis of MMP13, ADAMTS5 and COX2 expression level with LIPUS application for 20 min. C , D Western blot and quantitative analysis of P62, ATG5 and LC3 expression level with LIPUS application for 20 min. E , F Western blot and quantitative analysis of ACAN, COL2A1, SOX9 and COX2 expression level with 30 mW/cm 2 LIPUS application. G Western blot and quantitative analysis of ATG5 expression level with 30 mW/cm 2 LIPUS application. H qPCR of MMP3, ADAMTS5 and MMP13 relative expression level. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
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YAP exacerbated chondrocyte damage induced by IL-1β. Chondrocytes transfected with si-NC or YAP siRNA following IL-1β induction for 24 h. A qPCR of MMP3, MMP13 and ADAMTS5 relative expression level. B Western blot and quantitative analysis of COL2A1, MMP3, ADAMTS5 and MMP13 expression level. C , D Western blot and quantitative analysis of COX2, iNOS, P62 and LC3 expression level. E , F Immunofluorescence staining and fluorescence intensity analysis of COL2A1 and MMP13 relative expression level. G , H Chondrocytes were transfected with si-NC or si-YAP and then transfected with mRFP-GFP-LC3 adenovirus following IL-1β treatment for 24 h. The representative images of fluorescence and quantitative analysis of red dots and yellow dots were shown (scale bar: 10 μm). Subsequently, Chondrocytes transfected with oe-NC or oe-YAP following IL-1β induction for 24 h. I Western blot and quantitative analysis of COL2A1, MMP3, ADAMTS5 and MMP13 expression level. J , K Western blot and quantitative analysis of COX2, iNOS, Beclin-1 and P62 expression level. L – O Immunofluorescence staining and fluorescence intensity analysis of COL2A1 and MMP13 relative expression level. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
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The effect of mtDNA on IRF1 and chondrocyte damage. Chondrocytes were treated with TNF-α alone or CsA alone or TNF-α combined with CsA for 12 h. (A, B) Western blots and quantitative analysis of expression level of IRF1 ( n = 3). (C, D) immunofluorescence staining and fluorescence intensity analysis of IRF1 nuclear location ( n = 3). (E, F) Western blots and quantitative analysis of expression level of iNOS, MMP13, and MMP3 ( n = 3). Data are shown as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
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ZBP1 overexpression depends on IRF1. Chondrocytes were transfected with siNC or IRF1 siRNA for 24 h following TNF-α induction for 12 h. (A) Prediction of transcription factors using the UCSC Genome Browser database, SPP-ominer database, and Cistrome Data Browser. (B) qPCR results showing the relative mRNA expression levels of Irf1 and Zbp1 ( n = 3). (C, D) Western blots and quantitative analysis of the relative protein expression level of IRF1 ( n = 3). (E, F) Immunofluorescence staining and fluorescence intensity analysis of ZBP1 expression in chondrocytes transfected with siNC or IRF1 siRNA following TNF-α induction for 12 h ( n = 3; scale bar: 50 μm). (G-L) Western blots and quantitative analysis of the relative protein expression levels of iNOS, COX2, MMP3, MMP13, AGGRECAN, COL2A1, and SOX9 ( n = 3). (M-P) Immunofluorescence staining and fluorescence intensity analysis of COL2A1 and MMP13 expression in chondrocytes transfected with siNC or IRF1 siRNA following TNF-α induction for 12 h ( n = 3; scale bar: 50 μm). Chondrocytes were transfected with negative control siRNA(siNC) or IRF1 siRNA (si- Irf1 ) or ZBP1-plasmid (OE- Zbp1 ) or empty plasmid (OE-NC) for 24 h following TNF-α induction for 24 h. (Q-V) Western blots and quantitative analysis of the relative protein expression of AGGRECAN, COL2A1, SOX9, iNOS, COX2 MMP13, and MMP3 in chondrocytes ( n = 3). The data are shown as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
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ZBP1 is essential for chondrocyte damage. Chondrocytes were transfected with siNC or ZBP1 siRNA for 24 h following TNF-α induction for 24 h. (A, B) Western blots and quantitative analysis of ZBP1, MMP13, and MMP3 expression levels ( n = 3). (C, D) Western blots and quantitative analysis of SOX9 expression levels. (E, F) Western blot and quantitative analysis of iNOS expression levels ( n = 3). (G) qPCR results showing the relative expression levels of Zbp1 and Inos ( n = 3). (H-K) Immunofluorescence staining and fluorescence intensity analysis of the relative expression levels of COL2A1 and MMP13 ( n = 3; scale bar: 50 μm). Chondrocytes were transfected with siNC or ZBP1 siRNA following TSZ (20 ng/ml TNF-α, 100 nM Smac mimetic, and 20 mM Z-VAD) induction for 12 h. (L, M) Western blots and quantitative analysis of ZBP1, P-RIPK3, and P-MLKL expression levels ( n = 3). The data are shown as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
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Effects of MOIG on adhesion, migration, invasion and the expression of associated proteins of TNF-α-stimulated FLSs cells. (A) Adhesion of FLSs cells. (B) and (C) The wound healing of FLSs at 24 and 48 h; (D) and (F) The migration of FLSs at 6 h; (E) and (G) The invasion of FLSs at 24 h; ( H–M ) the expression of ICAM-1, VCAM-1, cadherin 11, MMP2 and MMP3 of FLSs. The data were expressed as mean ± SD (n = 3). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. normal ctrl group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α model group.
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ICCB-19 protects against TNF-α-induced detrimental events via autophagy mechanism. After a pre-treatment of ICCB-19 for 2 h, chondrocytes were stimulated by TNF-α for 24 h. A the protein expression of ATG5 and LC3 was examined. B The semi-quantitative analysis of protein bands by image J. C Representative images of immunofluorescence staining of LC3 in chondrocytes, scale bar: 50 μm. D – F After a pre-treatment of ICCB-19 and 3-MA for 2 h, chondrocytes were stimulated by TNF-α for 24 h with or without z-VAD, the protein expression of iNOS, COX2, COL2A1, MMP13, MMP3, C-caspase-9, and C-caspase-3 were examined, and the phosphorylation of RIPK1, RIPK3, and MLKL as well as protein expression of these three markers were detected by western blot. G – H After a pre-treatment of Rapa for 2 h, chondrocytes were stimulated by TNF-α for 24 h with or without z-VAD, the protein expression of iNOS, COX2, MMP13, C-caspase-3 and the phosphorylation of RIPK1, RIPK3, and MLKL as well as protein expression of these three markers were detected by western blot. I The graphical scheme summarized the findings of protective role of ICCB-19 in TNF-α-caused detrimental events. Data are expressed as means ± SD. * p < 0.05, ** p < 0.01. z-VAD, Z-VAD-FMK. C-caspase-9, cleaved-caspase-9. C-caspase-3, cleaved-caspase-3. P-, Phosphorylated-.
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LIPUS inhibits the progression of OA via YAP/RIPK1 axis. A Western blot and quantitative analysis of ACAN COL2A1, SOX9 and MMP3 expression level. B Western blot and quantitative analysis of COX2 and iNOS expression level. C Western blot of P-YAP and P-P65 expression level. D , E Immunofluorescence staining and fluorescence intensity analysis of COL2A1 and MMP13 relative expression level. F Chondrocytes were transfected with mRFP-GFP-LC3 adenovirus following IL-1β treatment for 24 h. The representative images of fluorescence and quantitative analysis of red dots and yellow dots were revealed (scale bar: 10 μm). G Western blot of P-YAP, YAP, P-P65, P65, P-IκB, IκB, P-RIPK1 and RIPK1 relative protein expression. H Images of IF staining for P65 relative expression and the distribution in chondrocytes with IL-1β induction for 15 min (scale bar: 25 μm). I Co-IP experiment of the binding between YAP and RIPK1 after LIPUS intervention. J YAP and RIPK1 colocalization in chondrocytes were detected by IF after LIPUS intervention (scale bar: 10 μm). K , L Immunohistochemistry staining to show the P-YAP-positive cell, P-RIPK1-positive cell and P-P65-positive cell in cartilage of the six groups (scale bar: 100 μm). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
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