Western Blot Analysis of Cell Signaling

Cell lysates were prepared as previously described [11 (link)]. Whole cell extract of 30 μg protein was resolved in SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk/PBS for 30 min and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study include anti-PDGFRA (Cell Signaling, 3174T), anti-COL1A1 (Proteintech, 67288-1-Ig), anti-ACTA2 (α-SMA, ABclonal, A7248), anti-SM22 (TAGLN, Proteintech, 10493-1-AP), anti-PROX1 (Boster, BA2390), anti-PDPN (Proteintech, 11629-1-AP), anti-VEGF-A (Immunoway, YT5108), anti-CD31 (Proteintech, 11265-1-AP), anti-VEGFR2 (Proteintech, 26415-1-AP), anti-VCAM (Proteintech, 11444-1-AP), anti-VEGFR3 (ABclonal, A5605), anti-ETAR (EDNRA, Santa Cruz, sc-135902), anti-ICAM (Proteintech, 10831-1-AP), and anti-β-actin(Sigma, A5441). Anti-IR Dye 800 or Dye 680 anti-rabbit or anti-mouse IgG antibodies (LI-COR Biosciences) were used as the secondary antibodies. An Odyssey system (LI-COR Biosciences) was used for the detection of proteins of interest.