Nupage sample reducing agent and sample buffer

siRNA-transfected ECs were seeded into FN-coated 10 cm dishes at a density of 2 × 10⁶ cells/dish, and incubated for 48 h at 37°C and 5% CO₂. ECs were then lysed on ice in lysis buffer as previously described by Valdembri et al. (2009) (link) in the presence of 1× Halt protease inhibitor cocktail (Thermo Scientific) and protein quantified using the DC BioRad assay. 100 μg protein from each sample was immunoprecipitated by incubation with protein-G Dynabeads^® (Invitrogen) coupled to a rabbit anti-NRP2 antibody (clone 3366, Cell Signaling Technology) on a rotator overnight at 4°C. Immunoprecipitated complexes were then washed 3× with lysis buffer + 1× Halt^TM protease inhibitor cocktail, and once in PBS, before being added to and boiled in NuPAGE sample reducing agent and sample buffer (Life Technologies) for Western blot analysis.

HUVECs were grown to 80–90% confluency in 10 cm dishes coated with 10 μg/ml FN in PBS. Cells were lysed in RIPA buffer. Four hundred micrograms of total protein from each sample was immunoprecipitated by incubation with protein G Dynabeads (Invitrogen) coupled to 1 μg mouse-anti-human VEGF-A antibody (VG-1, Abcam) on a rotator overnight at 4°C. Immunoprecipitated complexes were washed three times with 0.2 ml of RIPA buffer, and once in PBS, before being added to, and boiled in NuPAGE sample reducing agent and sample buffer (Life Technologies) for western blotting.