Figure EV4. Validation of astrocyte and microglial proteomic markers that increase across the ALS‐FTD spectrum.

1. Western blot analysis for the astrocytic, HEPACAM and GFAP, and microglia, MSN and TTP1, hub proteins (M6) across randomly selected cases from control (n = 5), ALS (n = 12), ALS/FTD (n = 6) and FTD (n = 7) groupings. Both HEPACAM and TPP1 display high molecular weight species (indicated by asterisk *), representing well‐established glycosylation events on these proteins, respectively (Golabek et al, 2003; Moh et al, 2005), which were included in the quantification. Western blot for GAPDH is provided as a loading control (bottom panel). Microglial and astrocyte markers are highlighted in blue and green, respectively.
2. Quantification was performed by ImageJ and statistical significance determined by ANOVA with Dunnett's post hoc test to compare each disease group to the control group (*P‐value < 0.05). Box plots depict mean (horizontal bars) and variance (25^th to 75^th percentiles), with whiskers extending to the last non‐outlier measurements, as shown.
3. Representative immunohistochemistry images of cortex and white matter regions of the frontal cortex from case 20 (ALS) and case 30 (ALS/FTD). GFAP and HEPACAM staining in cells of the cortical layers demonstrate staining intensity in astrocytes in the frontal cortex, whereas MSN and TPP1 staining show a microglial expression pattern. The relative increase in immunoreactivity for GFAP, HEPACAM, MSN, and TPP1 in the ALS/FTD case likely reflects changes in the abundance, of astrocytes and microglia measured by proteomics in module 6 (red). Scale bars represent 50 μm in low‐power images and 20 μm in insets.