Protein extraction was performed with ice‐cold RIPA buffer (Thermo) supplemented with a protease inhibitor cocktail followed by homogenization on ice and centrifugation at 14,000g for 15 minutes. Protein concentration in the supernatant was determined with Pierce bicinchoninic acid assay (Thermo). Laemmli buffer (Bio‐Rad) was prepared by adding 5% 2‐mercaptoethanol and used to dilute samples to 50 µg for loading. Proteins were denatured at 95°C for 5 minutes, and separated on Mini‐PROTEAN TGX Stain‐Free 4%–15% gradient gels (Bio‐Rad) for 30 minutes at 300 V, and transferred to nitrocellulose membranes with Trans‐Blot Turbo (Bio‐Rad).
Membranes were blocked for 30 minutes in 10% nonfat dry milk (NFDM) dissolved in TBST (Tris‐buffered saline + 0.1% Tween 20). A CD31 antibody (Abbiotec, San Diego, CA) or a VEGF‐A antibody (Abcam, ab46153, 1:500) was diluted 1:200 in 5% NFDM/TBST and incubated overnight at 4°C with gentle rocking. Membranes were washed (TBST), incubated with horseradish peroxidase goat anti‐Rabbit IgG antibody (1:3,000 in 5% NFDM/TBST) for 1 hour at RT, and then washed again. For visualization, membranes were activated and imaged using the ChemiDoc MP imaging system (Bio‐Rad). As opposed to the use of loading controls, stain‐Free total protein was used for normalization44 , 45 , 46 .
Membranes were blocked for 30 minutes in 10% nonfat dry milk (NFDM) dissolved in TBST (Tris‐buffered saline + 0.1% Tween 20). A CD31 antibody (Abbiotec, San Diego, CA) or a VEGF‐A antibody (Abcam, ab46153, 1:500) was diluted 1:200 in 5% NFDM/TBST and incubated overnight at 4°C with gentle rocking. Membranes were washed (TBST), incubated with horseradish peroxidase goat anti‐Rabbit IgG antibody (1:3,000 in 5% NFDM/TBST) for 1 hour at RT, and then washed again. For visualization, membranes were activated and imaged using the ChemiDoc MP imaging system (Bio‐Rad). As opposed to the use of loading controls, stain‐Free total protein was used for normalization