Fig 2: Dysregulated Pten expression in forebrains of Ptenm3m4/m3m4 mice correlates with decreases in synaptic marker expression and increased microglial activation in vivo.a Expression of Pten in the forebrain of PtenWT/WT, PtenWT/m3m4, and Ptenm3m4/m3m4 mice quantified by western blot analyses at P2, P8, P14, P21, and P40; P2 (relative expression = 0.63; H = 11.92, p value = 0.002), P8 (relative expression = 0.52; H = 11.70, p value = 0.002), P14 (relative expression = 0.69; H = 7.98, p value = 0.01), P21 (relative expression = 0.41; H = 14.24, p value = 0.0005), P40 (relative expression = 0.36; H = 12.44, p value = 0.001). b Expression of Syn in the forebrain of PtenWT/WT, PtenWT/m3m4, and Ptenm3m4/m3m4 mice quantified by western blot analyses at P2, P8, P14, P21, and P40; P40 (relative expression = 0.62; H = 10.43, p value = 0.02). c Expression of Psd-95 in the forebrain of PtenWT/WT, PtenWT/m3m4, and Ptenm3m4/m3m4 mice quantified by western blot analyses at P2, P8, P14, P21, and P40; P40 (relative expression = 0.60; H = 8.6, p value = 0.01). d Immunofluorescent staining for Syn (green) and Iba1 (red) in PtenWT/WT, PtenWT/m3m4, and Ptenm3m4/m3m4 CA3 hippocampal regions at P14 and P40. e Quantification of Syn in CA3 region of the hippocampus at P14 (mean integrated density of Syn: PtenWT/WT = 1.06 ± 0.13; PtenWT/m3m4 = 1.15 ± 0.46; F = 7.17, p value = 0.02; Ptenm3m4/m3m4 = 1.85 ± 0.41; F = 7.17, p value = 0.01) and P40 (mean integrated density Syn: PtenWT/WT = 0.96 ± 0.33; Ptenm3m4/m3m = 0.47 ± 0.04; F = 4.9, p value = 0.03), as well as microglia cell area P40 (mean Iba1 area: PtenWT/WT = 3.62 ± 0.89; Ptenm3m4/m3m4 = 10.41 ± 1.70; F = 12.5, p value = 0.0009).

Fig 3: Co-IP’d peptides and proteins identified comparing aCSF vs. KCl in WT lysates. (A) Volcano plot showing peptides that were enriched and depleted in aCSF vs. KCl. Peptides that were identified as enriched in WT vs KO in the aCSF condition are color-coded as shown in the key. (B) The same volcano lot from (A) is shown with the scale changed to highlight significantly depleted peptides, and all peptides that meet significance criteria color-coded by protein identity. Key shows color-coding of peptides by protein identify. (C) Volcano plot showing proteins that were depleted in aCSF vs. KCl. Depleted proteins are labeled. (D) StringDB node-edge diagram of the identified peptides from (B), highlighting the known interactions. (E) IP-western blots confirm that ARP3, PSD95 (DLG4) and SynGAP co-associate with Homer1, but not with nonspecific IgG control.

Fig 4: Workflow. a 3-mm sections of frontal cortex or bilateral hippocampi were collected from matched pairs of wildtype and mutant littermates. b P2 fractions were prepared to enrich for synaptic proteins. Shown here is typical enrichment of Homer1, PSD95, and NMDAR1 in P2 fractions, compared to equal amounts of total protein (by BCA assay) from brain homogenate (HO), P1 membrane pellet, and S2 soluble protein. c A panel of IP beads, each conjugated to a different antibody, is incubated with lysate, probed with fluorophore-conjugated antibodies, and read on a flow cytometer. d Known protein-protein interactions among the targeted protein network, in mouse, from the BioGRID database. Red lines indicate IP-western interactions, black lines IP-mass spectrometry. e, f Example histograms and corresponding node-edge visualizations. e Reduced IP: Shank3 Probe: Shank3 in a Shank3B-/- animal. Blue loop on Shank3 indicates a negative log2FC of an IP_Probe for the same target. f Increased Homer_PSD95 in VPA cortex lysate (red) and matched wildtype littermate control (black). Red line between nodes indicates positive log2FC of an interaction

Fig 5: Distribution of cortactin, and other pre- and postsynaptic markers at PC-PF synapses. (A) Graph showing the axo-dendritic distribution of gold particles coding for cortactin. Cortactin accumulated at the synapse, extending into the cytoplasmic fringe of the PSD. (See insets for explanation: orange shaded area is presynaptic, while blue is postsynaptic) (B) Axo-dendritic distribution of gold particles coding for synaptophysin, and PSD-95. Synaptophysin concentrated presynaptically, while PSD-95 was highly restricted to the postsynapse. (See insets for explanation) (C) Radial distribution of particles coding for cortactin. Radial distances of gold particles coding for cortactin from the closest membrane of spine heads were shown in the graph as proportions. Most cortactin particles were found in the immediate vicinity of the non-synaptic membrane. (D) Normalized distribution of cortactin shows that cortactin, independent of spine size, is restricted to the submembrane domain while particles from samples containing normal rabbit serum as control were found to be distributed more evenly within the spinoplasm. Data are from 195 profiles for cortactin, 45 for synaptophysin, and 45 for PSD-95. The bin width was 35 nm for (A), 20 nm for (B,C), and 0.05 normalized unit for (D). In D data were smoothed with a three-point weighted running average.