Figure 2.

Application of pSNAP to mouse primary cortical neuronal cultures

(A) Schematic representation of the pSNAP workflow. Mouse primary cortical neurons are pulse labeled for 2 h with a combination of 10 μM puromycin and heavy amino acids or only medium-heavy amino acids. After IP of NPCs with anti-puromycin antibodies, NPCs are eluted with 0.15% TFA and digested into tryptic peptides. The resulting peptide sample is analyzed by LC/MS/MS.

(B) Multi-scatter plots of log₂ H/M ratios from three independent experiments using mouse primary neurons.

(C) pSNAP can enrich NPCs from primary neurons. Relative starting positions of identified peptides within proteins. The bars represent averaged values from three independent experiments.

(D) Experimental design for the differential nascent proteome profiling of the mouse primary cultures between DIV 5 and 14.

(E) Representative images of primary neuronal cultures (DIV5 and DIV14) stained with DAPI (nuclear marker), NeuN (neuronal marker), MAP2 (dendritic microtubule marker), PSD95 (postsynaptic marker), and SYPH (presynaptic marker). Scale bar, 30 μm.

(F) A volcano plot showing differential NPC levels between DIV 5 and 14. Significantly regulated proteins were identified based on a combination of the t-test p-value (p < 0.05) of three replicates and the mean of the SILAC ratios [above 0.5 (a log₂ ratio)] (dash lines) which correspond to FDR <0.05 (see STAR Methods).

(G) Gene ontology enrichment analyses for the significantly upregulated NPCs (adjusted p < 0.01).