Fig. 3. Identification of the postsynaptic proteome of layer 5 neurons.

a Representation of L5 IT and PT neuron position and morphology in the somatosensory cortex with overview of input localization from other local and long-range excitatory neurons (adapted from Radnikow and Feldmeyer, 2018). b AAV-mediated Cre-dependent GFP expression in Tlx3-Cre+ and Sim1-Cre+ mouse lines recapitulates soma position for L5 IT and PT neurons, respectively. Layer 5 borders were identified by density of DAPI-positive nuclei. Scalebar is 200 μm. These experiments were independently repeated more than 3 times with similar results. c Workflow: mice are injected with AAVs in the somatosensory cortex at P28. At P35, biotin is provided 3 h before collection of somatodendritic material from somatosensory cortices. Biotinylated proteins are enriched by streptavidin pulldown and prepared for MS analysis. d MS replicates were collected from Tlx3-Cre+ and Sim1-Cre+ mice for no.turboID, cytosolic.turboID or PSD95.turboID conditions. e PCA analysis shows replicates clustering by condition but not by Cre line. f Statistical analysis of cytosolic.turboID and PSD95.turboID samples for each Cre line to determine synaptic protein enrichment. Core constituents of the excitatory postsynapse are annotated. Numbers of identified cytosolic and synaptic proteins for Tlx3-Cre+ and Sim1-Cre+ lines are indicated in bottom left and right corners of each graph. Two-sided, unpaired t-test with permutation-based FDR correction at 10%, s0 > 0.1. g Gene ontology analysis of the cytosolic and synaptic proteins from (f) showing cellular localization of identified proteins. VPM ventral posteromedial thalamic nucleus, POm posteromedial thalamic nucleus, IT intratelencephalic, PT pyramidal tract, PCA principal component analysis.