Fig. 3

ISG15 induction causes changes in neuronal EV protein content. A, B Mature cultures of human ISG15 targeted gene knockout or non-targeted control (NTC) neuronal cultures were treated with 100 ng/mL IFNγ for 24 h and then lysates were immunoprecipitated with anti-ISG15 (clone F9) and proteins were eluted in 5% glacial acetic acid for washing and LCMS analysis. Proteins with > 5 exclusive unique peptide counts (A) or total spectrum counts (B) and >5-fold enrichment in NTC precipitates compared to ISG15 KO precipitates were considered significant. Means and SEM are presented. C Primary mouse cortical neurons were transfected at DIV 0 with 2000 MOI of AAV1.Syn.shISG15-eGFP or AAV1.Syn.eGFP empty vector control. Media was fully replaced on DIV 7 and cells were treated every other day with an additional bolus of 100 ng/mL IFNγ, and EVs were isolated from supernatant 7 days later (DIV 14). Proteins were extracted from exosomal lysates and acquired by LCMS. Proteins with > 5 exclusive unique peptide counts (A) or total spectrum counts (B) and >5-fold enrichment in shISG15 transfected lysates compared to eGFP transfected controls were considered significant and exclusive unique peptide counts are presented as heat maps. Mean ± SEM are shown