Fig 5. Endogenous proximity labeling proteomics reveals the Pants interactome.

A) experimental design: A homomology-driven repair construct targeting the V5 epitope tag fused to the TurboID proximity labeling biotinylation enzyme followed by the self-cleaving t2A peptide and GFP to the 3’ end of the endogenous Pants locus was co-transfected with a vector carrying the sgRNA and Cas9 into Neuro2A cells. Single cells expressing GFP were then sorted into the wells of 96-well plates, generating clonal knock-in cell lines. B) Knock-in was verified by genotyping PCR of cell lines using a forward primer in the Pants gene and a reverse in GFP. C) Expression of the Pants-V5-TurboID fusion protein in cell lines was verified by Western blotting of cell lysates with an antibody to the V5 epitope. D) Treatment of Pants-Turbo cells with 50 μM biotin for 18 hours resulted in specific biotinylation of proteins not seen in N2A controls, as verified by Streptavidin-HRP immunoblotting. E) Pie chart showing localization pattern of Pants targets identified by proximity-labeling proteomics. F) IPA analysis of functional linkage between unique proteomic hits, showing SZ connections and inter-gene connections. G) Proteins uniquely biotinylated in Pants-TurboID cell line, ordered by Sequest Score. Those implicated in plasticity, memory or cognitive disease are highlighted in red. H) Validation of proteomics results by biotinylation followed by streptavidin pulldown and Western blot with selected antibodies specific to Pants targets identified by Mass spectrometry.