Ab21269

Near-confluent HMVEC WT and CMG2 / TEM8 KD cells were washed three times in cold PBS and lysed for 30 min at 4°C in PBS supplemented with 1% Triton X-100 and a protease inhibitor cocktail (cOmplete mini protease inhibitor cocktail, Roche). Lysates were cleared by centrifugation at 16,000 xg and the supernatant protein concentration was measured by BCA assay. Protein (40μg) from each sample was used for SDS-PAGE and blotted onto PVDF. Following blocking, the membranes were probed with antibodies against CMG2 (1:1000 16723–1-AP, Proteintech, Rosemont, IL) or TEM8 (1:1000 ab21269, Abcam, Cambridge, UK), then stripped and reprobed for beta-actin (1:10,000 A5441, Sigma) as loading control. Blots were imaged using a ChemiDoc imager (Bio-Rad, Hercules, CA).

The 70–80% confluent cell lysates were collected using M-PER® buffer supplemented with protease and phosphatase inhibitor cocktail (Roche Applied- Science). Protein concentrations were measured by Bradford assay (Thermo Scientific). Lysates containing 10–30 μg of protein were electrophoresed using 10% SDS-bispolyacrylamide gels, and separated proteins in the gels were transferred to nitrocellulose membranes. After 1 hour blocking in 5% milk/TBST, the membranes were incubated overnight at 4 °C with primary antibodies diluted in 4% BSA (Sigma Aldrich)/TBST solution. After washing, the membranes were incubated with secondary antibodies for 1 h at room temperature and immunoreactive bands were visualized by chemiluminescent substrate (Thermo Scientific). Secondary antibodies, anti-mouse HRP (1:1000, #32430, Thermo Scientific), anti-rabbit HRP (1:10,000, #31460, Thermo Scientific) and anti-goat HRP(1:5000, sc-2056, Santa Cruz)were diluted in 5% milk/TBST solution. The following primary antibodies were used for western blotting: ANTXR1 (1:1000, ab21269, Abcam), Flk1 (1:1000, sc-504, Santa Cruz), p-Flk1 (1:1000, sc-101820, Santa Cruz), Flt1 (1:1000, sc-316, Santa Cruz), VEGF-A (1:500, sc-152, Santa Crus), and β-actin (1:5000, A5441, Sigma Aldrich) and HA (1:500, sc-805, Santa Cruz).