Pdgfrβ fc

400 ng of each of pcDNA3-gH (full-length), pcDNA3-gL-FLAG and pcDNA3-gO-8his were transfected in to Expi293F cells at 2 × 10⁶ / ml using Expifectamine. Transfection Enhancers were added 22 h later, and cells were harvested 46 h post-transfection. Cells were washed with PBS-BSA and incubated with PDGFRα-Fc (purified as described) or PDGFRβ-Fc (R&D Systems) for 40 minutes on ice. Cells were then washed 3 times with PBS-BSA and stained with anti-FLAG M2-Cy3 (Sigma), chicken anti-HIS-FITC (polyclonal, Immunology Consultants Laboratory), and anti-human IgG-APC (clone HP6017, BioLegend) for 30 minutes on ice. Cells were washed three times with PBS-BSA and analyzed by flow cytometry. To compare data across different experiments, the change in ΔMFU for each condition was normalized to the ΔMFU at the maximum concentration of WT sPDGFRα-Fc: Relative binding = (MFU_sample−MFU_background) / (MFU_{max WT} − MFU_background).

PDGFRβ-expressing pericytes were incubated with FAM- or IR700-labeled Z_PDGFRβ affibody at room temperature for 1 h. After two washes with PBS, the cells were analyzed by using a flow cytometer. To block the binding of Z_PDGFRβ affibody to PDGFRβ, cells were pre-incubated with a goat anti-human PDGFRβ antibody for 1 h before the addition of the Z_PDGFRβ affibody. In addition, PDGFRβ-binding of the Z_PDGFRβ affibody was further determined by using protein interaction analysis performed on an OpenSPR system (Nicoya Life Sciences Inc., Kitchener, Canada) (McGurn et al., 2016 (link)). Briefly, PDGFRβ-Fc (R&D, MN) was immobilized on the COOH-sensor chips, and solutions containing increasing concentrations of the Z_PDGFRβ affibody were introduced onto the chip, followed by surface plasmon resonance analysis on the OpenSPR system. The kinetic constants, including the association constant (K_a), dissociation constant (K_d) and affinity (KD, KD = K_d/K_a), were calculated using software according to a 1:1 binding model.