Ab13822

Cells were homogenized in either Radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher) supplemented with protease inhibitor mixture (Pierce), or directly in 1× Laemmli buffer (Bio-rad, Hercules, CA, USA) supplemented with β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations of RIPA lysates were determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher) with BSA as a standard. Proteins (20–40 μg) were separated on a run on 4-20% polyacrylamide Tris-glycine gels (Thermo Fisher) and transferred to polyvinylidene difluoride (PVDF) membranes. The transferred membranes were blocked for 1 hour at room temperature in Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA) and incubated at 4°C overnight with primary antibodies against: human and rhesus PECAM1 (1:500; JC/70A, Abcam, Cambridge, MA, USA), human and mouse VE-cadherin (1:200, C-19, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), phospho-AKT (Ser473) (1:1000, 12694S, Cell Signaling, Danvers, MA, USA), phospho-ERK1/2 (Thr202/Tyr204) (1:1000, 4370, Cell Signaling), α-tubulin (1:5000, ab89984, Abcam), Vinculin (1:1000, V4139, Sigma), and β-actin (1:1000, ab13822, Abcam). The signals were visualized with an Odyssey imaging system.