Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Flow cytometry sorting of the combinatorial Scl library. (A) Library expression was monitored by staining with a phycoerythrin‐conjugated antibody binding to a primary anti‐c‐Myc antibody. The purple rectangle gate indicates cells with the highest expression. (B) The library was incubated with 5 n m LRP4, and the low‐affinity library fraction was collected (purple triangle). (C) The low‐affinity library was incubated with 650 n m LRP6, and the purple rectangle represents clones that bind to LRP6.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Clone Assay

Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Identification of affinity‐reducing mutations. Heat maps demonstrating significantly enriched Scl variants in (A) LRP4 LOW library compared to Scl NAIVE library fractions; (B) LRP4 LOW LRP6 library compared to LRP4 LOW library fractions; (C) LRP4 LOW library compared to Scl NAIVE library fractions that overlap with the LRP4 LOW LRP6 library. The heat maps present the log 2 transformation of the ER (red scale bar on the right‐hand side) and highlight single mutations that significantly (A) reduce the binding affinity to LRP4, (B) reduce the binding affinity to LRP4 and retain binding to LRP6, and (C) overlap in (A) and (B). The substituting amino acids are shown on the X ‐axis, and the substituted positions are shown on the Y ‐axis. Statistical significance was determined by a two‐sided Poisson exact test and multi‐test corrected by the Benjamini–Hochberg FDR.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Transformation Assay, Binding Assay

Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Scl single‐mutation variants selected for empirical validation by YSD.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Biomarker Discovery, Variant Assay

Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: YSD binding of Scl WT and the selected single‐mutation variants to LRP4. Geometric mean fluorescence intensity (Geo MFI) is presented as a fold change. Recombinant yeast cells expressing Scl WT or its variants were incubated with (A) 1 n m , (B) 10 n m , or (C) 50 n m soluble LRP4. The binding signal of each Scl variant was normalized first to the expression signal of the corresponding variant and then to the binding signal of Scl WT at the respective LRP4 concentration. Each experiment was repeated at least three times, and the results are presented as means ± SD. Statistical significance was assessed using an unpaired, two‐tailed Student's t ‐test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Binding Assay, Mutagenesis, Fluorescence, Recombinant, Expressing, Incubation, Variant Assay, Concentration Assay, Two Tailed Test

Journal: Febs Letters

Article Title: Mapping the sclerostin– LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: SPR analysis of binding of purified Scl WT and Scl single‐mutation variants to LRP4. SPR data showing binding of (A) Scl WT , (B) Scl K75Q , (C) Scl K75E , and (D) Scl V136D to 3 μg of immobilized LRP4 receptor. Different protein concentrations are represented by different colors.

Article Snippet: For library sorting, the cells were incubated with 5 n m soluble His‐tagged human LRP4 or 650 n m soluble His‐tagged mouse LRP6 (R&D Systems, Minneapolis, MN, USA) and 1 : 100 affinity‐purified chicken anti‐c‐Myc antibody (Immunology Consultants Laboratory, Inc., Portland, OR, USA) for 1 h at room temperature, followed by double staining with a 1 : 50 FITC‐conjugated anti‐6 × His‐tag monoclonal antibody (Invitrogen, Waltham, MA, USA) and 1 : 50 Alexa FluorTM 555‐labeled goat anti‐chicken IgY (H + L) antibody (Invitrogen) for 20 min in the dark on ice.

Techniques: Binding Assay, Purification, Mutagenesis