A construct corresponding to In-PDGFRα (DNAFORM, AK035501, RIKEN clone 9530057A20) was obtained. This construct was subcloned into the pMXs-IRES-GFP retroviral backbone (Cell BioLabs, Inc.) to generate pMXs-I-Pα. Replication-incompetent retroviral particles were generated by transfection of the 293T human embryonic kidney cell-derived Phoenix helper cell line (gift from Dr. Gary Nolan, Stanford University). Viral supernatant was filtered through 0.45 μm polyethersulfone filters, concentrated using PEG precipitation, and stored at −80°C.
FAPs were plated in 6-well plates and grown in DMEM supplemented with 10% FBS. When cells reached 70% confluency, viral supernatant and polybrene (at a final concentration of 4 μg/ml) were added to the medium. For overexpression experiments, FAPs were incubated with the viral supernatant for 48 hours before analysis. For signaling assays, FAPs were incubated with the viral supernatant for 24 hours. Afterwards, the medium was then changed to serum-free DMEM containing viral supernatant and the cells were incubated for an additional 24 hours. The FAPs were then treated with 1 ng/ml PDGF-AA for 15 minutes, after which the cells were used for Western blot analysis.
FAPs were plated in 6-well plates and grown in DMEM supplemented with 10% FBS. When cells reached 70% confluency, viral supernatant and polybrene (at a final concentration of 4 μg/ml) were added to the medium. For overexpression experiments, FAPs were incubated with the viral supernatant for 48 hours before analysis. For signaling assays, FAPs were incubated with the viral supernatant for 24 hours. Afterwards, the medium was then changed to serum-free DMEM containing viral supernatant and the cells were incubated for an additional 24 hours. The FAPs were then treated with 1 ng/ml PDGF-AA for 15 minutes, after which the cells were used for Western blot analysis.