Identification of PDGFR-β Activation

PDGFR-β activation was identified and tested using the following methods: receptor tyrosine kinase (RTK) array, Western blotting, and MesoScale analysis.

Cell lines were prepared, lysed, and compared according to the protocol from the Proteome Profiler Human Phospho-RTK Array Kit (Bio-Techne, Minneapolis, MN).

Cells were plated in RPMI growth medium for 24 hours. After 24 hours, medium was removed, and each plate was treated with ligand stimulation with 25 ng/ml of PDGF-BB (Bio-Techne) or vehicle in RPMI with glutamine only. Plates were incubated for 10 minutes at 37°C and then immediately placed on ice for harvest. Cells were washed twice with ice-cold PBS (Life Technologies) and then lysed with LDS lysis buffer (Sigma Aldrich) with phosphatase and protease inhibitors (Life Technologies).

Protein lysates (30-60 μg/lane), as determined by BCA protein assay (Life Technologies), were separated in 4% to 12% SDS-PAGE (Life Technologies) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were blocked with 5% nonfat dried milk in TBS (KPL, Gaithersburg, MD)-Tween 20 (Sigma Aldrich) (20 mm Tris-HCI, pH 7.5; 8 g/l of sodium chloride; 0.1% Tween 20). Blots were incubated with antibodies against total PDGFR-β and phospho-PDGFR-β (tyr751) (Cell Signaling Technology, Beverly, MA) at a 1:1000 dilution. Antiactin antibody from Abcam Inc. (Cambridge, MA) was used as a loading control. Bands were visualized on camera using West Femto ECL detection reagent (Life Technologies).

Protein lysates were quantified by BCA protein assay (Life Technologies) and added to MesoScale Discovery Multi-spot 4 Spot 96-well plates (Rockville, MD). Samples were analyzed in triplicate using the MesoScale Discovery Sector Imager 2400 plate reader.