Summary of antibody performance for ALS-RAP proteins.

(A) Overview of the identification of high-quality antibodies targeting ALS-RAP proteins. (B) Percentage of ALS-RAP targets covered by at least one specific renewable (monoclonal or recombinant) antibody for the indicated application. (C) Percentage of tested antibodies that met specificity criteria in each indicated application.

YCharOS antibody characterization workflow for Galectin-1 (LGALS1).

Ten Galectin-1 antibodies were evaluated side-by-side across three applications using HAP1 WT and LGALS1 KO cells. (A) Whole-cell protein lysates from HAP1 WT and LGALS1 KO cells (20 µg per lane) were analyzed in WB using the indicated Galectin-1 antibodies. Ponceau-stained membranes are shown to assess protein loading and transfer efficiency. (B) Whole-cell protein lysates (10 µg) and conditioned media (10 µg) from HAP1 WT and LGALS1 KO cells were analyzed in WB using antibody 12936, as in (A), to detect intracellular and secreted Galectin-1. (C) Immunoprecipitation was performed from HAP1 WT cell lysates using the indicated Galectin-1 antibodies. Starting material (SM), unbound fractions (UB), and whole immunoprecipitates (IP) were analyzed by WB. Anti-Galectin-1 antibody A21218 was used as a primary antibody. (D) HAP1 WT and LGALS1 KO cells were differentially labeled with green and far-red fluorescent dyes, respectively, mixed at a 1:1 ratio, and plated in optically clear 96-well plates. Cells were stained with the indicated Galectin-1 antibodies, followed by Coralite 555–conjugated secondary antibodies and DAPI. Images were acquired in the blue (DAPI), green (WT), red (antibody staining), and far-red (KO) channels. Representative grayscale images of the blue and red channels are shown; WT and KO cells are outlined with green and magenta dashed lines, respectively. Scale bar, 10 µm. Recombinant antibodies are denoted by double asterisks (**) and monoclonal antibodies by single asterisks (*) after the catalogue numbers; polyclonal antibodies are unmarked. Antibody screening was performed in two independent experiments; representative data from one experiment are shown.

Accessing curated antibody characterization data through the OGA website.

YCharOS antibody characterization data for ALS-RAP proteins are accessible through the Only Good Antibodies (OGA) website (https://onlygoodantibodies.co.uk/). The OGA homepage provides a searchable interface for the 33 ALS-RAP proteins, together with 35 additional neurological disease–associated proteins evaluated by YCharOS, and includes links to key OGA publications, data interpretation guidelines, antibody characterization protocols, and a portal for nominating new targets. (A) Representative results page generated by searching LGALS1, showing filters for host species, clonality, recombinant format, and OGA-recommended applications; the WB filter is applied. A schematic panel at the top illustrates ideal antibody performance across applications, while antibodies matching the selected filters are displayed in randomized order. (B) Number of antibodies returned following application of the selected filters.

Detection of 30 ALS-RAP proteins across human-derived neurological cell types.

WB showing detection of 30 ALS-RAP proteins across the indicated cell types. For each protein, two independent experiments are shown: chemiluminescence-based WB (left), performed using a defined lysate batch across all corresponding blots, and fluorescence-based WB (right), performed using a second independent lysate batch. Lysates were prepared from the indicated human iPSC-derived cell types and primary cells. Proteins are listed in alphabetical order. A representative Ponceau-stained membrane and total protein stain are shown to assess protein loading and transfer efficiency. Vertical lines between the motor neuron and DA neuron lanes indicate that the blots were spliced to remove additional conditions not relevant to this study. In (A), eight lysates were used across both detection modalities. In (B), the fetal astrocyte lysate was not available for either detection modality. In (C), the fetal astrocyte lysate was not included in the chemiluminescence-based experiment. Lane identities: 1, motor neurons; 2, DA neurons; 3, oligodendrocytes; 4, astrocytes; 5, microglia, 6, fetal astrocytes; 7, fetal microglia; 8, MDMs. DA, dopaminergic; MDM, monocyte-derived macrophages. (D) Summary of 16 targets for which WB data and Human Protein Atlas RNA expression profiles are concordant, grouped by expression category.

Characterization of ALS-RAP targets.

Number of PubMed entries related to ALS-RAP genes. Black points represent the number of entries retrieved using the search term “gene name + ALS,” while gray points represent entries retrieved using the gene name alone. The x-axis indicates the number of PubMed entries, and the y-axis lists the gene names.

ALS-RAP RNA expression across brain cell types.

Heatmap showing normalized RNA expression values from the Human Protein Atlas (HPA) single nuclei brain dataset. Expression is given as nTPM values on a scale from 0-500. Genes denoted with an asterisk (*) are classified as “barely detectable” but show substantial expression in a single cell type.

Protein-level characterization of iPSC-derived neurological cells.

(A) Table listing cell type–specific marker proteins. (B) WB analysis of marker proteins using antibodies against the indicated proteins. A Ponceau-stained membrane is shown to assess protein loading and transfer efficiency. Lysates were prepared from the indicated human iPSC-derived cell types or fetal astrocyte culture. Vertical lines between the motor neuron and DA neuron lanes and between the DA neuron and microglia lanes indicate that WB were spliced to remove additional conditions not relevant to this study. DA, dopaminergic; NPC, neuronal progenitor cells.

Quality control and marker validation of iPSC-derived microglia.

(A) Immunofluorescence staining showing expression of IBA1 and PU.1 in iMGs, with no detectable signal in undifferentiated iPSCs. (B) Representative flow cytometry plots demonstrating expression of CD45 and CD11b in iMGs but not in iPSCs. (C) Quantification of the percentage of CD45-positive, CD11b-positive, and double-positive cells in iPSCs and iMGs, as measured by flow cytometry (N = 4). Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test. (D) RT-qPCR expression levels of canonical microglial markers in iMGs compared to iPSCs (N = 3). Statistical analysis was performed using Student’s t-test. iMG, iPSC-derived microglia; iPSC, induced pluripotent stem cell. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bar, 50 µm

STRING analysis of known physical interactions between ALS-RAP proteins.