Screen of AIM-binding nanobodies by yeast surface display. (A) Schematic of the constructed yeast display library. Genes of VHH nanobodies were fused to that encoding the Aga2p subunit and VHH proteins were displayed on the yeast surface. (B) Schematic of the screening strategy. Yeasts were sorted for AIM-A1 binding, amplified, depleted with A1-CAIM or NAIM-A1 and re-sorted for AIM-A1 binding to obtain AIM-specific binders. Each protein is marked by the starting and ending VWF residues. (C-G) Flow cytometry plots showing binding of yeasts to AIM-A1 after various screening steps. In each plot, y-axis shows FLAG-tagged nanobody-Aga2p fusion protein expression on the yeast surface that is detected by Alexa Fluor 488-conjugated anti-FLAG antibody, and x-axis shows binding of the biotinylated AIM-A1 protein as detected by streptavidin-conjugated allophycocyanin. NAIM- and CAIM-specific binders were isolated from the approximate gates illustrated in the plots (D,F). After the second enrichment, individual clones (E,G) were isolated, amplified, and confirmed for AIM-A1 binding.