Figure 5.

Quantitative Proteomics Analysis of Arc^TAP Reveals a Depletion of Postsynaptic Proteins in PSD95 Knockout Mice

(A) Arc complexes were isolated from Arc^TAP/+ and Arc^TAP/TAP mice crossed with PSD95 knockout mice (Arc^TAP/TAPxPSD95^−/−) by FLAG capture and Tev protease release (single-step purification). Total lysate (IN, input) and the same volume of lysate upon purification (SN) and Tev elution from both genotypes were blotted against Arc and quantified. Eluted Arc levels following the FLAG capture from Arc^TAP/+ and Arc^TAP/TAPxPSD95^−/− lysates were not statistically different (Mann-Whitney U test, p = 0.1). Data are presented as mean ± SEM.

(B) Isolated complexes from (A) were resolved by SDS-PAGE and stained with colloidal Coomassie. Three independent purifications are shown. The lanes were cut for LC-MS/MS analysis, and the identified proteins are listed in Table S7. TAP-tagged Arc, PSD95, and the Tev enzyme are indicated.

(C) Dimethyl labeling-based quantitative MS of TAP-purified proteins from Arc^TAP/+ and Arc^TAP/TAP crossed with PSD95 knockout mouse forebrain (Arc^TAP/TAP×PSD95^−/−). The plot displays enrichment ratios of Arc^TAP/TAP×PSD95^−/− versus Arc^TAP/+ (x axis) and iBAQ enrichment values of the step purification (y axis). Proteins meeting criteria for enrichment (>1.5 fold) are highlighted in green and for depletion (< 0.667 fold) are highlighted in red. The names of depleted and enriched PSD95 interactors are indicated. See the Supplemental Experimental Procedures for enrichment criteria.

(D) Mouse interactome network constructed from the publicly available databases BioGrid, Database of Interacting Proteins (DIP), IntAct, Molecular INTeraction Database (MINT), STRING database, UniProt, Biomolecular Interaction Network Database (BIND) and mentha using the Psicquic software package. The network is visualized using Visone. Proteins highlighted in green/red meet the enrichment/depletion criteria discussed in the Supplemental Experimental Procedures.