fibulin 3  (R&D Systems)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

doi: 10.1016/j.mcpro.2022.100223

Figure Lengend Snippet: ADAMTS7 candidate substrates identified in all TAILS experiments

Article Snippet: To validate EFEMP1 cleavage in a binary assay, purified recombinant human HA-tagged EFEMP1/Fibulin-3 (R&D, 8416-FB) provided in PBS was dialyzed into TBS pH 8.0 to prevent precipitation with the CaCl 2 in the assay buffer.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

doi: 10.1016/j.mcpro.2022.100223

Figure Lengend Snippet: Validation of TAILS substrate EFEMP1 and cleavage site preference. A , EFEMP1/Fibulin-3 protein domains, amino acid sequence of the atypical EGF repeat linker, and location of the ADAMTS7 cleavage sites. Abbreviated EFEMP1 domains: signal peptide (SP), N-terminal region (N), EGF repeats (E). B , concentrated medium from HUVEC expressing Ad-Luc, Ad-mWT, or Ad-mEQ assessed by Western blot under nonreducing conditions. Anti-EFEMP1 antibody recognizes an epitope C-terminal to the ADAMTS7 cleavage sites. C , quantitation of semi-tryptic or semi-chymotryptic peptides from HUVEC medium matching novel cleavage sites from the endogenous EFEMP1 protein. The total area was greater for the 123.124 cleavage site compared to the adjacent 124.125 cleavage site. Additional cleavage events observed were also found in the Luc and EQ controls. D , in vitro cleavage of HA-EFEMP1 by purified full-length mouse ADAMTS7 S3A assessed by Western blot. The antibodies to the N-terminal HA epitope and C-terminal EFEMP1 epitope recognized the EFEMP1 more strongly under nonreducing conditions. A band at 100 kDa under nonreducing conditions is consistent with a purified HA-EFEMP1 dimer, which was also sensitive to ADAMTS7 cleavage. E , overnight digest of HA-EFEMP1 by mouse ADAMTS7 S3A assessed by Coomassie staining. F , quantitation of semi-tryptic or semi-chymotryptic peptides from the atypical EGF1 repeat region from HA-EFEMP1, showing a consistent preference for the 123.124 cleavage site. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; E, EGF repeats; HUVEC, Human umbilical vein endothelial cells; N, N-terminal region; SP, signal peptides; TAILS, terminal amine isotopic labeling of substrates.

Article Snippet: To validate EFEMP1 cleavage in a binary assay, purified recombinant human HA-tagged EFEMP1/Fibulin-3 (R&D, 8416-FB) provided in PBS was dialyzed into TBS pH 8.0 to prevent precipitation with the CaCl 2 in the assay buffer.

Techniques: Sequencing, Expressing, Western Blot, Quantitation Assay, In Vitro, Purification, Staining, Isotopic Labeling

Journal: Frontiers in Pharmacology

Article Title: EFEMP1 Overexpression Contributes to Neovascularization in Age-Related Macular Degeneration

doi: 10.3389/fphar.2020.547436

Figure Lengend Snippet: Serum fibulin-3 levels in wet AMD-affected patients and controls. Fibulin-3 serum concentration in wet AMD-affected patients was significantly higher than that in the controls. ** p < 0.01.

Article Snippet: In some cases, WT or shEFEMP1 HUVECs were preincubated in ECM with different concentrations of recombinant human fibulin-3 protein (8416-FB-050, R&D Systems, Minneapolis, MN).

Techniques: Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: EFEMP1 Overexpression Contributes to Neovascularization in Age-Related Macular Degeneration

doi: 10.3389/fphar.2020.547436

Figure Lengend Snippet: Origin of the proangiogenesis property of EFEMP1. (A) Tube formation of wild type (WT) HUVECs treated with different concentrations (0 µg/ml, 1 µg/ml, 2 µg/ml, 4 µg/ml, and 8 µg/ml) of recombinant human fibulin-3 protein and shEFEMP1 HUVECs treated with or without fibulin-3 protein was tested. (B) Proliferation of wild type (WT) HUVECs treated with different concentrations (0 µg/ml, 1 µg/ml, 2 µg/ml, 4 µg/ml, and 8 µg/ml) of recombinant human fibulin-3 protein, WT HUVECs in fibulin-3 coated or uncoated plates, and shEFEMP1 HUVECs treated with or without fibulin-3 protein was tested using EdU assay. # p > 0.05.

Article Snippet: In some cases, WT or shEFEMP1 HUVECs were preincubated in ECM with different concentrations of recombinant human fibulin-3 protein (8416-FB-050, R&D Systems, Minneapolis, MN).

Techniques: Recombinant, EdU Assay