Phospho-antibody microarray anayses for control and DG-LRG1 treated HUVEC cells.

Deglycosylated-leucine-rich α-2-glycoprotein1 (DG-LRG1) as well as LRG1 was discovered to promote angiogenesis under diabetes mellitus condition through TGF-β independent binding to endoglin. To examine the signaling pathways triggered by DG-LRG1, we subjected whole-cell protein lysates of control and DG-LRG1 treated HUVECs to a Phospho Explorer antibody array analysis using commercial antibody array assay kit (Full Moon Biosystems, Inc.). Samples were probed against 1318 site-specific and phospho-specific antibodies with two replicates per antibody printed on a coated glass microscope slide. Phospho Explorer antibody array experiments and analyses were performed as a custom service by E-Biogen (Ebiogen Inc., Seoul, Republic of Korea). HUVECs were serum-starved for 8 hours and then incubated with an anti-TGFβ1 antibody for 1 hour, after which HUVECs were stimulated with 1μg/ml DG-LRG1 for 30 minutes. Whole-cell lysates of control and DG-LRG1 treated HUVECs were prepared, and aliquots of lysates containing 50 μg of protein were used for Phospho Explorer antibody assays (Full Moon Biosystems, Inc.).