Fig 1: miR-147b attenuates LPS-induced endothelial hyperpermeability to albumin in HLMECs by targeting ADAM15.(A) Representative Western blot from human lung microvascular endothelial cells (HLMEC) transfected with 200 nM miR-147b mimic or a miR negative control. (B) ADAM15 expression was measured 48 h post-transfection and quantified with densitometry. (C) Incubation with 200 ng/ml LPS for 24 h caused an increase in albumin permeability across HLMEC monolayers, an effect diminished in cells transfected with miR-147b. Data represent mean ± SEM. *P<0.05 vs. control scrambled miR, # P<0.05 vs. LPS scrambled miR.
Fig 2: miR-147b down-regulates cell surface expression of ADAM15 in endothelial cells.At 48 h after transfection, live HUVECs were labeled with a monoclonal anti-ADAM15 ectodomain antibody and a secondary Cy3 conjugate. Cell surface expression was analyzed by flow cytometry (A–B). The bar graph shows quantitative cell surface ADAM15 expression in different treatment groups, each derived from 3 separate experiments (C). Data represent mean ± SEM. * P<0.05 vs. untreated control.
Fig 3: miR-147b down-regulates ADAM15 expression in endothelial cells.(A) Representative Western blot from HUVECs transfected with 200 nM miR-147b mimic or a scrambled miR control. (B) Quantitative densitometric analysis of ADAM15 expression at 48 h post-transfection. (C) Representative Western blot from HUVECs transfected with 200 nM miR-147b antagomir or a control antagomir. (D) Quantitative densitometric analysis of ADAM15 expression at 48 h post-transfection. Data represent mean ± SEM. *P<0.05 vs. scrambled control.
Fig 4: miR-147b targets ADAM15 3′ UTR.(A) Schematic illustration of ADAM15 mRNA and sequence alignments between ADAM15 3′ UTR and hsa-miR-147b. (B) HUVECs were co-transfected with a miR mimic and pmirGlo/ADAM15 containing ADAM15 3′ UTR downstream of the luciferase reporter. Cells were analyzed for luciferase activity 48 h post-transfection. Data represent mean ± SEM. *P<0.05 vs. scrambled control.
Fig 5: miR-147b attenuates LPS-induced increase in ADAM15 expression.Immunofluorescence labeling of ADAM15 in HUVECs transfected with scrambled miR (A), miR-147b mimic (B), scrambled miR and 200 ng/ml LPS (C), and miR-147b mimic and 200 ng/ml LPS (D). Green denotes ADAM15 staining, blue is DAPI staining of nuclei. Scale in (A) corresponds to 20 µm and applies to all images.
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