Extended Data Fig. 9: Validation of DELTA in the PSD-95-HaloTag mouse.

a, Experimental procedures for testing saturation in a PSD-95–HT mouse. b Example section showing the basal dendrite section of the CA1 region of the hippocampus (DAPI shows the nuclei in the leftmost panel). The green channel (autofluorescence; 2^nd panel from left) accounts for all the signal in the red channel (Chase – JF₅₅₂-HTL; 3^rd panel), while the far-red channel shows the expected pattern of synapses (Pulse – JFX₆₇₃-HTL; 4^th panel). These results were consistnat across n = 3 mice tested. c, Validation of HTL signal (JF₅₅₂-HTL; middle panel) in the PSD-95-HT knock-in animal with a knockout validated antibody (left panel) showing high colocalization (right panel). Repeated for 4 fields of view in 1 animal. d, Box plot of all pairs of animals (6 animals, n = 15 pairs) for correlation coefficients for the lifetime of PSD-95 across 12 large brain regions (left panel; As in Fig. 2g, i) or cortical layers and HC subfields (right; As in Fig. 2f, j). Shown are the median (center), interquartile range (bounds of the box), and whiskers representing 1.5× the interquartile range. e, Violin plot of coefficient of variation (CV) across animals for the total PSD-95 measured for each brain region. Pulse only is publicly available data from Bulovaite et al.²⁹ where we used the standard deviation divided by the mean of integrated fluorescence at day 0 (n = 7 animals; n = 111 brain regions). For DELTA we used the standard deviation divided by the mean of the total Pulse and Chase values (n = 6 animals; n = 466 brain regions). Measurement variability of total PSD-95 across animals was lower in DELTA then in Bulovaite et al. (two-sided Wilcoxon rank sum test; z = 12.6; p = 1.3e-36). Shown are also the median (circle), interquartile range (bounds of the box), and whiskers representing 1.5× the interquartile range. f-g, Correlation between estimated expression of PSD-95 (y axis) with its lifetime measurement (x axis) from data of Bulovaite et al. (f; Pulse only) or from DELTA (g) for twelve brain regions (colored circles). r² values from linear regression are shown for each panel. See Supplementary Text for details. h, Workflow for mass spectrometry (MS) measurements of protein turnover. i, Comparisons of PSD-95 mean lifetime with or without HaloTag fusion by different methods. There is an agreement between DELTA and MS based measurements of lifetime for PSD-95-HT. The non-overlapping confidence intervals of the pulse-only approach signifies a statistically significant difference. n = 4 mice for MS; Same data as Fig. 2g for DELTA; Pulse only data from ref# 29. j, Volcano plots comparing MS-based proteome-wide lifetime estimates between PSD-95-HaloTag knock-in mice and WT cage mates. Left, per protein analysis showing most proteins don’t significantly change in lifetime (y-axis: -log₁₀ p value – two-sided; horizontal line at p = 0.05; x-axis: log₂ fold change; vertical lines at two-fold increase or decrease). PSD-95 is highlighted with a red square; significant single proteins are labeled with their gene names. Right, same as left but averaging proteins based on their GO Cellular Component annotations. Synapse Cellular Component is highlighted with a red square. No correction was made for multiple comparisons.