Fig 1: PEG10 binds to RNAs and RNA-binding proteins, localizes to stress granules and extracellular vesicles (EVs) in AS(A) IP-MS analysis of PEG10 binding partners in AS versus AS + PEG10 KD using 2 PEG10-specific antibodies (Ab 1, Ab 2). n = 4 independent IPs, values in heatmap represent average log10 label-free quantification (LFQ) intensities from MaxQuant; hits selected are enriched in PEG10 IPs with an adjusted p value cutoff of 0.001. NA, saline treatment; P10, PEG10 KD.(B) Validation of ATXN2 and ATXN10 binding to PEG10 in AS by IP-immunoblotting.(C) Representative immunostainings for PEG10-RF1-GFP and RF1/2-GFP expressed in H4 cells co-stained with G3BP1 after PBS treatment or treatment with 0.5 mM sodium arsenite (45 min; scale bar: 20 µm).(D) Representative immunostainings for PEG10-RF1/2 in AS neurons along with ATXN2 and MAP2 upon PBS or 0.5 mM sodium arsenate treatment (45 min; scale bar: 20 µm).(E) Left: volcano plot (adjusted p value versus log2 FC AS versus control) of PEG10 RNA immunoprecipitation sequencing (RIP-seq) in control and AS neurons representing the 1,000 most abundant transcripts in PEG10 IPs. Horizontal line represents adjusted p value cutoff of 0.05. Vertical line represents FC > 0. Right: the 20 most abundant transcripts in PEG10 IPs. Data are expressed as raw counts per million and are represented in descending order. Stars represent statistically significant enrichment in AS neurons versus control, adj. p < 0.05.(F) Schematic of EV isolation from human iPS-derived neurons.(G) Representative transmission electron microscope (TEM) images after uranyl acetate negative staining (magnification: 15,000×; scale bar: 200 nm).(H) Hydrodynamic diameter (Dh) of vesicles isolated from control and AS neurons analyzed by dynamic light scattering. Results are shown as means ± SEMs, n = 3 independent replicates for each line.(I) EV particle concentration (per mg EV protein) measurements from control and AS EVs analyzed by multi-angle dynamic light scattering (MALDS). Results are shown as means ± SEMs, n = 3 independent replicates from each line.(J) Representative immuno-EM measurements for PEG10-RF1/2 and TSG101 in EVs from AS cells (magnification: 15,000×; insert 4× magnification; scale bar: 200 nm).(K) Quantification of PEG10-RF1/2-positive EVs from control and AS cells (n = 3 independent EV preparations, p value: Mann-Whitney test).(L) LC-MS heatmap for PEG10 and its binding proteins and selected EV markers in control and AS lysates (values are protein-level intensities obtained from Spectronaut and are averages of 3 independent lysate and EV preparations).(M) Immunoblotting analysis for PEG10-RF1/2 and ATXN10 along with EV markers with equal total protein loaded for lysates and EVs (for quantification, see Figure S5D).