Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/eLife.42274

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Article Snippet: Peptide, recombinant protein , recombinant human pro-Collagen I α1 , R and D Systems , 6220 CL , .

Techniques: Recombinant, Diagnostic Assay, Cell Culture, Protease Inhibitor, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Fractionation

Journal: JCI Insight

Article Title: PhIP-Seq uncovers marked heterogeneity in acute rheumatic fever autoantibodies

doi: 10.1172/jci.insight.196619

Figure Lengend Snippet: ( A ) Volcano plot of differential autoantibody reactivity (PhIP-Seq peptides) between ARF cases and healthy controls. Significant peptides (using DESeq2, adjusted P < 0.05) colored by average binding magnitude, nonsignificant by sample density. Number of peptides significantly elevated in ARF cases compares with healthy controls is indicated; 118 peptides mapping to 81 proteins. Dashed lines indicate thresholds for significance and no change; enriched targets PPP1R12B and COL1A1 labeled. Total magnitude indicated by red scale; ARF significantly elevated magnitude compared with healthy controls by 1-sided Wilcoxon’s test. P = 0.018, r = 0.35. ( B ) GO pathway enrichment for ARF-associated autoantigens ( P < 0.01, 81 proteins represented by 118 peptides in A ). Dot size reflects protein count per pathway; fill color indicates enrichment strength. Outlines denote GO categories: Cellular Component (blue), Biological Process (green). Highlighted pathways include Sarcomere and Heart Morphogenesis. Full pathway analysis results with enrichment metrics for all GO terms are provided in . ( C ) Heart muscle expression of ARF-enriched targets (Human Protein Atlas). Upper panel: RNA expression ordered from low to high; tile borders indicate multiple (green) or single (gray) peptide hits. Lower panel: Protein expression by immunohistochemistry; tile borders indicate staining reliability (enhanced, red; supported, orange; approved, pink; uncertain, gray). Red arrows highlight PPP1R12B and COL1A1. ( D ) Subcellular localization and secretion prediction (Human Protein Atlas). Upper panel: Localization across cellular compartments (purple = presence); borders show reliability. Lower panel: Secreted proteins prediction (violet = predicted secreted). Main locations shaded if proteins predicted secreted as main location. PPP1R12B and COL1A1 indicated by red arrows.

Article Snippet: Recombinant antigens were either obtained commercially — human COL1A1 ( NM_000088 , Origene) and PPP1R12B ( NM_032104 , Origene) — or produced recombinantly using Expi293 cells as the expression host and purified using standard nickel affinity and size exclusion chromatography (CD226; NM_006566 , Callaghan Innovation).

Techniques: Binding Assay, Labeling, Immunopeptidomics, Expressing, RNA Expression, Immunohistochemistry, Staining

Journal: JCI Insight

Article Title: PhIP-Seq uncovers marked heterogeneity in acute rheumatic fever autoantibodies

doi: 10.1172/jci.insight.196619

Figure Lengend Snippet: ( A ) Top: Schematic of PhIP-Seq peptide tiling for PPP1R12B protein; significant peptides (green) cluster at C-terminus. Middle: Heatmap of normalized, bead-corrected enrichment across PPP1R12B for ARF cases (purple) and controls (gold), clustered by donor profiles. Bottom: Mean enrichment for ARF and controls. ( B ) Individual peptide enrichment for PPP1R12B peptides in ARF (purple) versus controls (gold); significantly enriched peptides (17A, 18B, 19A) highlighted green with P values from 2 analysis shown in black text above. ( C ) Correlation of enriched PPP1R12B peptides in ARF. Upper panels: Pearson’s correlation coefficient ( r ) with P values from the regression show in red text. Lower panels: Scatterplots with regression lines and R 2 values. ( D ) Enrichment of COL1A1 peptides in ARF (purple) and controls (gold). ( E ) Correlation analysis between COL1A1 peptides in ARF. Upper panel: Pearson’s correlation coefficient ( r ) with P values from the regression shown in red text. Lower panels: scatterplots with regression lines and R 2 values. Box-and-whisker plots show median (line in box), interquartile range (IQR, box bounds), and 1.5 × IQR (whiskers).

Article Snippet: Recombinant antigens were either obtained commercially — human COL1A1 ( NM_000088 , Origene) and PPP1R12B ( NM_032104 , Origene) — or produced recombinantly using Expi293 cells as the expression host and purified using standard nickel affinity and size exclusion chromatography (CD226; NM_006566 , Callaghan Innovation).

Techniques: Whisker Assay

Journal: JCI Insight

Article Title: PhIP-Seq uncovers marked heterogeneity in acute rheumatic fever autoantibodies

doi: 10.1172/jci.insight.196619

Figure Lengend Snippet: ( A ) Schematic representation of the PPP1R12B PhIP-Seq peptide tiling and design of synthetic peptides used for validation. The 3 overlapping PhIP-Seq peptides significantly enriched in ARF (17A, 18B, 19A) localize to a contiguous region of the protein. Two nonoverlapping 45-mer peptides (Pep-1 and Pep-2) were synthesized spanning this region, with Pep-2 fully contained within the recombinant protein used for whole-protein ELISA. Created with Biorender. ( B ) Whole-protein ELISA responses to PPP1R12B, COL1A1, and CD226 in the discovery cohort (ARF vs. healthy). Violin plots with points; white circle symbols show the median, with IQR as error bars. Horizontal dashed lined indicate the reference threshold of 2 times the median of healthy controls. ( C ) Median absorbance in PPP1R12B peptide ELISAs in the discovery cohort; dashed lines indicate the reference threshold of 2 times the median of healthy controls. Wilcoxon’s P values (ARF vs. healthy) are shown per peptide. ( D ) Spearman’s correlation of PhIP-Seq normalized enrichment values for peptide 18B with ELISA absorbance values for PPP1R12B whole protein and Pep-2 with fitted linear regression lines (black) and Spearman’s correlation coefficient with significance shown in black text. ( E ) Discovery ROC for Pep-2 on the absorbance scale (ARF vs. healthy). The prespecified operating cutoff is marked in red with dotted guides. AUC and 95% CI are shown on the plot. ( F ) Independent validation cohort (ARF, healthy, and Strep A–positive pharyngitis controls): distribution of Pep-2 absorbance with the prespecified discovery cutoff (red dashed line). Wilcoxon’s P values are shown throughout in black text.

Article Snippet: Recombinant antigens were either obtained commercially — human COL1A1 ( NM_000088 , Origene) and PPP1R12B ( NM_032104 , Origene) — or produced recombinantly using Expi293 cells as the expression host and purified using standard nickel affinity and size exclusion chromatography (CD226; NM_006566 , Callaghan Innovation).

Techniques: Biomarker Discovery, Synthesized, Recombinant, Enzyme-linked Immunosorbent Assay