Fig. 5: Functional activity of FCRL5 and PDGFRα binding proteins.

a HeLa cells engineered to express FCRL5 (HeLa-FCRL5) receptor in a Dox-inducible manner were treated with 50 nM neutravidin-labeled pHrodo DeepRed complexed FCRL5bp, and live cell imaging (top panels) was used to measure overlap of GFP (FCRL5-expressing cells) and pHrodo Red fluorescence (internalized FCRL5bp). 1000 ng/mL doxycycline results in strong GFP expression and intracellular pH activated pHrodo Red fluorescence. Internalization (measured arbitrary units calculated by overlap of red and green fluorescence divided by phase area) reached steady state in 18 hours (images are from this timepoint); values are presented as mean ± SD for n = 3 replicate wells. b, c EGFR is degraded HeLa-FCRL5 cells by de novo binders. Measuring EGFR degradation by immunostaining and Western blot (b) or flow cytometry (c); n = 3 independent biological replicates for western blots and flow cytometry. FCRL5bp fusion to EGFRbp resulted in comparable degradation to the native degradation mechanism (EGF signaling). Gating strategies for flow cytometry are provided in Supplementary Fig. 9. d, e Western blot analysis of PDGFRα inhibition in Chinese Hamster Ovary cells engineered to overexpress PDGFRα (repeated four times with similar results). Levels of phosphorylated PDGFRα, Erk, and Akt were measured by immunoblots with fluorescent antibodies. Signals were normalized by the fluorescent signal of an antibody against the constitutively expressed housekeeping proteins S6 or actin. Data are presented as mean ± SD of n = 3 independent biological replicates. Source data are provided as a Source Data file.