Rabbit ve cadherin

Protein samples were loaded on 8% or 12% polyacrylamide gels and run until the appropriate protein separation was achieved. Samples were transferred onto polyvinylidene difluoride (PVDF) membrane and blocked for 1 h in 5% nonfat milk in TBST (Tris-buffered saline + 0.1% Tween 20). The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; Oh and Gu, 2013b (link)), goat anti-SH3BP1 (Everest Biotech Ltd.; Parrini et al., 2011 (link); Elbediwy et al., 2012 (link)), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich). The intensity of individual bands was quantified using ImageJ.

For in vitro experiments, olaparib (Selleckchem, Planegg, Germany) was used at 5 μm for 24 h. For in vivo experiments, olaparib (MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in DMSO and further diluted in 10% (2‐hydroxypropyl)‐β‐cyclodextrin (Sigma Aldrich, Darmstadt, Germany) in PBS. olaparib was administered intraperitoneally at a dose of 50 mg/kg. Treatment with olaparib or vehicle took place three times a week for 3 weeks.
Antibodies and dilutions for tissue immunofluorescence staining were rat CD34 (BD553731; BD Biosciences, Franklin Lakes, NJ, USA), 1:25; rabbit VE‐cadherin (ab205336; Abcam, Cambridge, UK), 1:500; mouse S100B (S2532; Sigma Aldrich), 1:100; mouse SMA‐Cy3 (C6198; Sigma Aldrich), 1:200; donkey anti‐rat immunoglobulin (Jackson Immunoresearch, Ely, UK), 1:200; donkey anti‐rabbit immunoglobulin (Jackson Immunoresearch), 1:200; and horse anti‐mouse immunoglobulin (Vector Laboratories, Burlingame, CA, USA), 1:100.