Journal: JCI insight

Article Title: A collagen IV-derived peptide disrupts α5β1 integrin and potentiates Ang2/Tie2 signaling.

doi: 10.1172/jci.insight.122043

Figure Lengend Snippet: Figure 5. AXT107 influences cell-cell junctions through Tie2-associated mechanisms. (A) Representative Western blot images of lysates from MECs treated with 200 ng/ml Ang2 and various concentrations of AXT107 targeting phospho-MLC2, with GAPDH as a loading control. (B) Densitometric analysis of images in A (n = 2 or 3). ***P < 0.001 by 1-way ANOVA relative to Ang2-alone control. (C) Representative immunofluorescence images of HUVEC mono- layers treated with 200 ng/ml Ang2, negative control, or Tie2 siRNA, and DMSO or AXT107 that have been stained with DAPI (blue) and antibodies against VE-cadherin (green) and F-actin (red). Scale bars: 25 μm. (D) Ratios of actual perimeters to minimal perimeters for images related to C. Ratios presented as averages from at least 3 cells from 3 different wells and 2 separate experiments (total of 5 values). ***P < 0.001 by 2-tailed Student’s t test.

Article Snippet: The cells were then blocked in blocking buffer (5% normal goat serum, 0.3% Triton X-100 in DPBS containing Ca2+ and Mg2+) and stained for 16 hours with primary antibodies against phospho-Tie2 (Y992) (R&D Systems, catalog AF2720), total Tie2 (Genetex, catalog GTX107505), VE-cadherin (Cell Signaling Technology, catalog 2500), or ZO-1 (ThermoFisher Scientific, catalog 33-9100) diluted 1:150 in antibody dilution buffer (1% BSA; 0.3% Triton X-100 in DPBS containing Ca2+ and Mg2+).

Techniques: Western Blot, Control, Immunofluorescence, Negative Control, Staining