Journal: British journal of cancer

Article Title: Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases.

doi: 10.1038/sj.bjc.6605850

Figure Lengend Snippet: Figure 7 Knockdown of WASF3 expression suppresses MMP9 expression levels and inhibits MMP9 release from prostate cancer cell lines. (A) RT–PCR analysis of PC3 and DU145 cells showing knockdown of WASF3 also show down-regulation of MMP9, but not MMP2 expression levels. Cont ¼ parental cells. (B) Secretion of the pro- and active form of MMP9 from PC3 and DU145 cells were measured in the supernatants of actively growing cells using the Fluorokine MAP assay procedure. Cells from each clone showing knockdown of WASF3 also showed reduced MMP9 levels compared with parental (control) and non-knockdown cells that did not.

Article Snippet: After 24 h, the media were collected in tubes and centrifuged for 10 min at 10 000 g. The pro- and active MMP-9 levels released into the media were measured using a Fluorokine MAP human MMP9 kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Antibodies

Article Title: Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody

doi: 10.3390/antib12010009

Figure Lengend Snippet: Activation of MMP9 by distinct proteases in vitro. Western blots for pro-MMP9 (92 kDa, red) and active MMP9 (84 kDa, green) after incubation with active ( A ) MMP7, ( B ) MMP12, ( C ) pepsin A, and ( D ) plasmin. Below each lane is the MMP9 activity towards gelatin expressed as gelatin fluorescence (arbitrary units) per minute. DQ, dye-quenched; MMP, matrix metalloproteinase.

Article Snippet: Sandwich immunoassays were conducted by coating the plate in 1 μg/mL recombinant antibody, adding recombinant MMP9 standard ADX, detecting with total MMP9 antibody (MAB936; R&D Systems, Minneapolis, MN, USA), and quantifying with a ruthenium-conjugated anti-mouse antibody (Meso Scale Discovery, Rockville, MD, USA).

Techniques: Activation Assay, In Vitro, Western Blot, Incubation, Activity Assay, Fluorescence

Journal: Antibodies

Article Title: Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody

doi: 10.3390/antib12010009

Figure Lengend Snippet: Discovery and characterization of antibodies directed at the active MMP9 neoepitope. ( A ) Hybridoma supernatants specific for the N-terminus of MMP9 after cleavage between Arg106 and Phe107 were identified by direct ELISA toward the corresponding on-target and off-target peptides. ( B ) Pro-MMP9 and active-MMP9 protein standards in buffer or cell lysate were probed with candidate hybridoma supernatants by Western blot. The multiple bands represent the multiple degradation products of MMP9. ( C ) Antibodies that reacted with FFPE cell pellets engineered to contain the active MMP9 neoepitope were identified. Orange stars indicate point mutations, either at the signal peptide junction with F107 (D20A or D20 deletion engineered to generate the F107 neoepitope; see ) or a mutation in the catalytic domain rendering MMP9 inactive. PDGFR TM and LDLR TM refer to transmembrane domains of PDGFR and LDLR, respectively. MMP3 G100L is an active form of MMP3, a negative control. Molecular weights of bands shown correspond to . ( D ) Specificity of the candidate IHC antibodies were determined using cell pellets in which the pro-MMP9:active MMP9 ratio was titrated using marimastat (0.025–2.5 μM). Inset Western blot images fall between the 98 and 62 kDa molecular weight markers. ( E ) A sandwich ELISA using anti–active MMP9 antibodies paired with total MMP9 antibody detected active MMP9 in diluent with a sensitivity <1 pg/mL. ( F ) ADX does not interfere with the sandwich ELISA for active MMP9. ADX, andecaliximab; BSA, bovine serum albumin; ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay; FFPE, formalin-fixed/paraffin-embedded; IHC, immunohistochemistry; LDLR, low-density lipoprotein receptor; MMP, matrix metalloproteinase; OD, optical density; PDGFR, platelet-derived growth factor receptor; PBS, phosphate-buffered saline; TM, transmembrane; WB, Western blot.

Article Snippet: Sandwich immunoassays were conducted by coating the plate in 1 μg/mL recombinant antibody, adding recombinant MMP9 standard ADX, detecting with total MMP9 antibody (MAB936; R&D Systems, Minneapolis, MN, USA), and quantifying with a ruthenium-conjugated anti-mouse antibody (Meso Scale Discovery, Rockville, MD, USA).

Techniques: Direct ELISA, Western Blot, Mutagenesis, Negative Control, Molecular Weight, Sandwich ELISA, Electrochemiluminescence, Enzyme-linked Immunosorbent Assay, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Derivative Assay, Saline

Journal: Antibodies

Article Title: Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody

doi: 10.3390/antib12010009

Figure Lengend Snippet: Pro- and active MMP9 in human IBD specimens. ( A ) Homogenized colon tissue from patients with ulcerative colitis and Crohn’s disease contain distinct levels of pro-MMP9 and active MMP9. ( B ) Pro-MMP9 is widespread in diseased colon tissue. ( C ) F107-MMP9 is focal and associated with diseased blood vessels and ( D ) fistulae. ( E ) Serum from patients with ulcerative colitis and Crohn’s disease contains higher levels of pro-MMP9 and active MMP9 than serum from healthy control donors. IBD, inflammatory bowel disease; MMP, matrix metalloproteinase; NETs, neutrophil extracellular traps.

Article Snippet: Sandwich immunoassays were conducted by coating the plate in 1 μg/mL recombinant antibody, adding recombinant MMP9 standard ADX, detecting with total MMP9 antibody (MAB936; R&D Systems, Minneapolis, MN, USA), and quantifying with a ruthenium-conjugated anti-mouse antibody (Meso Scale Discovery, Rockville, MD, USA).

Techniques: Control

Journal: Antibodies

Article Title: Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody

doi: 10.3390/antib12010009

Figure Lengend Snippet: Pro- and active MMP9 in hidradenitis skin specimens. ( A ) Pro-MMP9 is widespread in the skin of patients with hidradenitis, while active MMP9 is more focal and associated with ruptured epidermis (top) and ruptured hair follicles (bottom). ( B ) Active MMP9 and myeloid cells (MPO+ neutrophils and IBA1+ macrophages) are found proximal to the rupture in the skin, while lymphocytes (CD20+ B cells and CD3+ T cells) are secondary. F, fistula; IBA1, anti-ionized calcium-binding adaptor protein-1; MMP, matrix metalloproteinase; MPO, myeloperoxidase.

Article Snippet: Sandwich immunoassays were conducted by coating the plate in 1 μg/mL recombinant antibody, adding recombinant MMP9 standard ADX, detecting with total MMP9 antibody (MAB936; R&D Systems, Minneapolis, MN, USA), and quantifying with a ruthenium-conjugated anti-mouse antibody (Meso Scale Discovery, Rockville, MD, USA).

Techniques: Binding Assay