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.