Fig. 3: The axon initial segment proteome across development.

a Experimental design to study the AIS proteome during development using two parallel 10-plex TMT. b Pearson correlation showing high reproducibility in anti-NFASC targeted AIS proximity labeling for DIV7 cortical neurons (1532 common elements from 2 replicates). c Ranking DIV7 AIS biotinylated 1407 proteins according to their averaged MS intensity ratios (+ NFASC/+NeuN and +NFASC/ + MAP2). d DIV7 overall rank plot listing potential AIS proteins. e Pearson correlation showing high reproducibility in anti-NFASC targeted AIS proximity labeling for DIV21 cortical neurons (1859 common elements from 2 replicates). f Ranking DIV21 AIS biotinylated 1738 proteins according to their averaged MS intensity ratios (+ NFASC/+NeuN and +NFASC/ + MAP2). g DIV21 overall rank plot listing potential AIS proteins. h Venn diagrams showing 549 common AIS candidates at DIV7, 14 and 21. The prior AIS proteome at DIV14 was also included in this analysis. i Six clusters with distinct protein expression profiles were generated by pheatmap analysis along neuronal development. j The percentage of protein numbers in each cluster among 534 analyzed proteins. k AIS protein expression patterns along neuronal development. Proteins are represented by their gene names. l–n Volcano plots showing fold changes in AIS proteins between DIV7 and 14 (l), DIV 14 and 21 (m), or DIV7 and 21 (n). Red and blue points represent AIS proteins with significant up-regulation and down-regulation, respectively. Proteins are represented by their gene names. Horizontal dashed lines indicate p = 0.05, and unpaired two-tailed t test was used for p-value calculation. Vertical dashed lines indicate the cutoff of log₂(fold change) = ±1.