Extended Data Figure 8:

Library sorting process for non-LIFR binders. (a) Representative flow cytometry dot plot showing yeast library sorting process: isolation of non-LIFR binders (Sort 2 with 1 nM LIFR-Fc), isolation of CLCF1 binders (Sort 3 with 0.5 nM CLCF1-His), and isolation of non-LIFR binders (Sort 4 with 10 nM LIFR-Fc). To retain the binding affinity for CLCF1, strategy was alternated between positive screening for 0.5 nM CLCF1 and negative screening for increasing concentrations of LIFR-Fc. (b) Flow cytometry histogram representing yeast-displayed CNTFR variant containing T268A and D269A (CNTFR_AA) binding to CLCF1 (10 nM), gp130-Fc (10 nM), and LIFR-fc (10 nM) relative to untreated negative control (red). CNTFR_AA binds to CLCF1 and LIFR but not to gp130. For gp130-Fc and LIFR-Fc binding studies 10 nM of CLCF1 was added to induce receptor complex formation. All experiments were repeated at least three times with similar results.