Supplementary MaterialsS1 Fig: Amino acidity sequences of M-CSFWT, M-CSFRGD libraries, M-CSFc-FMS, M-CSFv3 as well as the 3 M-CSFRGD variants which were chosen following an affinity maturation process. c-FMS. (E) M-CSFc-FMS was made by changing the RGD motif on M-CSFRGD version 4.22 to RDG with desire to to avoid binding to v3 integrin. (F) Sequences from the mutated loop from the three M-CSFRGD clones which were chosen after four (4.22 and 4.24) and five (5.6) rounds from the affinity maturation procedure. M-CSF, macrophage ZM-447439 supplier colony-stimulating element; RGD, Arginine-Glycine-Aspartic acidity; WT, crazy type.(TIF) pbio.2002979.s001.tif (249K) GUID:?DA159A2C-589D-419E-A9CE-80AFD5C286D7 S2 Fig: Compatibility of YSD with M-CSFC31S. YSD M-CSFC31S was examined for (A) ahead scatter and part scatter and (B) manifestation using mouse anti-c-myc antibody accompanied by a second PE-labeled anti mouse antibody. (C) The binding of YSD M-CSFC31S to soluble c-FMS-Fc was recognized with a goat anti-human Fc-FITC antibody. (D) Cells expressing M-CSFC31S for the candida cell wall had been incubated with 10 different concentrations of c-FMS-Fc (0.5C2000 nM) and were tested for binding by movement cytometry. The curve displays a good healthy to an individual binding-site curve, as well as the obvious KD can be 20 nM. Resource data are available in S7 Data. FITC, fluorescein isothiocyanate; M-CSF, macrophage colony-stimulating element; Npy PE, phycoerythrin; YSD, candida surface screen.(TIF) pbio.2002979.s002.tif (707K) GUID:?6863EEE4-AAE6-4A57-9950-2DDA91155301 S3 Fig: Structure of YSD ZM-447439 supplier construct. The M-CSFRGD collection was covalently associated with Aga1p as well as the candida cell wall structure. Binding for c-FMS was determined with c-FMS-Fc recombinant protein and goat anti-human Fc FITC conjugated secondary antibody, and the expression levels were measured with a mouse anti-c-myc primary antibody and PE anti-mouse secondary antibody. For determination of v3 integrin binding, yeast cells were incubated with recombinant v3 integrin and mouse anti-human CD49d FITC secondary antibody, and the expression levels were measured with chicken anti-c-myc primary antibody and PE goat anti-chicken secondary antibody. FITC, fluorescein isothiocyanate; M-CSF, macrophage colony-stimulating factor; PE, phycoerythrin; RGD, Arginine-Glycine-Aspartic acid; YSD, yeast surface display.(TIF) pbio.2002979.s003.tif (378K) GUID:?8EBE58C7-3035-4DA8-BEF2-9252843CD354 S4 Fig: FACS dot plot of M-CSFRGD libraries. ZM-447439 supplier M-CSFRGD (ACD) library 1 and (ECH) library 2 were analyzed for (A and E) FSC/SSC, (B and F) expression, (C and G) 100 nM c-FMS binding, and (D and H) 500 nM v3 integrin binding. FACS, fluorescence-activated cell sorting; FSC, forward scatter; M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid; SSC, side scatter.(TIF) pbio.2002979.s004.tif (1.4M) GUID:?1D649EE8-6284-43C2-9134-4363A0A7F324 S5 Fig: FACS FSC and SSC of affinity maturation process. Yeast-displayed mutant libraries were analyzed, and the living cells population in each sort is represented by a black polygon-shaped gate. The affinity maturation sorting process started with (A) a presorted library followed by (B) sort 1, (C) sort 2, (D) sort 3, (E) sort 4, and (F) sort 5. FACS, fluorescence-activated cell sorting; FSC, forward scatter; SSC, side scatter.(TIF) pbio.2002979.s005.tif (473K) GUID:?E9C9DF75-B503-4820-A5B0-92D7E85EFE72 S6 Fig: Analysis of individual YSD M-CSFRGD clones selected from sorts 4 and 5 for their binding to c-FMS, v3 integrin ZM-447439 supplier and other integrins. Twenty-five different clones from each of sorts 4 (A) and 5 (C) were tested for binding to 20 nM of v3 integrin, normalized to the lowest binder. (B) The best 15 v3 integrin M-CSFRGD binders from sort 4 and the best 10 v3 integrin M-CSFRGD binders from sort 5 (D) were examined for binding to 50 nM of c-FMS, normalized to M-CSFC31S. The selected clones (4.22, 4.24, and 5.6) are indicated in blue. (E) Variations 4.22, 4.24, and 5.6 were evaluated for integrin specificity by tests their binding to 250 nM of 47, IIb3, v5, and 51 integrins in comparison to their binding to v3 integrin. Resource data are available in.