Immunoblotting for Gal1 and VEGFR2

Immunoblotting was performed as described (34 (link)). Briefly, equal amounts of rGal1 or protein extracts from HUVECs (30 µg) exposed to plasma samples preincubated for 1 h with human anti-Gal1 mAbs (clone 21 or 42) or isotype control were resuspended in Laemmli buffer under reducing conditions. Proteins were resolved in 4 to 20% SDS–PAGE and blotted onto nitrocellulose membranes (GE Healthcare). After blocking, the membranes were probed with anti-human Gal1 mAb-21 and mAb-42 (1.5 μg/mL): anti-VEGFR2 (1/1,000; Cell Signaling), anti-phospoho-VEGFR2 (1:1,000; Cell Signaling), anti-ERK-1/2 (1:2,000; Sigma), and anti-phospho-ERK-1/2 (1:1,000; Sigma). Blots were then incubated with horseradish peroxidase (HRP)–labeled anti-human, rabbit, or mouse secondary Ab 1:2,000 (Vector) and developed using the Immobilon Western Chemiluminescent HRP Substrate (Millipore). Protein bands were analyzed with Fiji 2.0 analysis software.