Fig. 4. APEX-based comparison between the nuclear and cytosolic proteomes of dSPNs and iSPNs.

a Targeted expression of APEX in direct and indirect-pathway spiny projection neurons (dSPNs and iSPNs). Some schematics were created with BioRender.com. b Western blot analysis of biotinylated striatal lysates prepared from Drd1^Cre and A2a^Cre mouse lines. Western blot analysis of these samples was performed once prior to orthogonal confirmation by proteomics. c Cre-dependent APEX-NES expression in the striatum of A2a^Cre mouse line. Left, sagittal section (1 mm) of A2a^Cre > 2 weeks post AAV transduction. Middle, soma-fill EGFP reporter in the striatum (STR) (100 µm). Right, iSPN-specific axonal projections in the external globus pallidus (GPe) (200 µm). n = 2 animals per construct. Similar EGFP expression pattern was confirmed for all A2a^Cre+ animals used in the proteomic experiment. n = 3 and 5 for NES and H2B, respectively. d Subcellular localization of APEX constructs in striatal iSPNs. Confocal images of APEX-expressing neurons including EGFP, immunostained FLAG-Ab, and streptavidin for visualizing APEX subcellular localization and biotinylation patterns (20 µm). n = 2 animals per construct. e Statistical analysis workflow for comparing dSPN and iSPN proteomes. Some schematics were created with BioRender.com. f Gene ontology analysis of the 458 H2B-enriched nuclear proteome generated from H2B – NES comparison. Non-H2B enriched proteins are considered as a part of the cytosolic proteome. g Differential expression analysis for the non-H2B enriched cytosolic proteome (A2a^Cre.NES – Drd1^Cre.NES). h Differential expression analysis for the H2B-enriched nuclear proteome (A2a^Cre.H2B – Drd1^Cre.H2B). i Workflow for correlation analysis between proteins and transcripts. j Correlation between log2FC (dSPN – iSPN) for proteins and translating mRNA TRAPseq dataset. k Same as (j) for mRNA expression from single-cell RNAseq dataset. Source data are provided as a Source Data file.