Pdgfrβ antibody clone y92

Lung pericytes were lysed with ice-cold RIPA lysis buffer (Cell Signaling, Danvers, MA). All lysed samples were kept on ice for 30 min and centrifuged for 10 min at 4°C at 12,000 × g. Cell lysates were subjected to 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 7% milk in TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for 1h. After washing with TBST twice, membranes were incubated with primary antibody overnight at 4°C. A rabbit monoclonal PDGFRβ antibody (clone Y92; Abcam), a rabbit polyclonal NG2 antibody (H-300; Santa Cruz Biotechnology) and a mouse monoclonal SV40T-antigen antibody (PAb416; Abcam) were used. Primary antibodies to β-actin and α-tubulin were obtained from Cell Signaling. Fli-1 primary antibody was provided by Dr. Xiankui Zhang (Medical University of South Carolina). The membranes were washed twice with TBST and incubated with HRP conjugated secondary antibody in blocking buffer for 1h. After washing three times with TBST, immunoreactive bands were visualized by incubation with ECL plus detection reagents (GE Healthcare, Chicago, IL). The densitometry of bands was quantified with Image J2 software.

HEK293 cells, normal and immortalized human lung pericytes grown on 12-well plates coated with 0.2% gelatin were fixed with 4% PFA for 15 min at room temperature and then washed with DPBS. After blocking with 1% BSA for 15 min at room temperature, cells were incubated overnight at 4°C with a rabbit monoclonal PDGFR-β antibody (clone Y92; Abcam, Cambridge MA) or a rabbit polyclonal NG2 antibody (H-300; Santa Cruz Biotechnology, Dallas, Texas). Cells were washed with DPBS and incubated for 1h at room temperature with Alexa Fluor 594 goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, Carlsbad, CA), diluted 1:200 in DPBS. After washing with DPBS, images were acquired using a Zeiss Observer.Z1 microscope with Zen 2 software.