Figure 1.

Identification and validation of p140Cap interactors. Enrichment of synaptic markers in the synaptosomal preparation. (A) Validation of the synaptosomes. Western blot for p140Cap, PSD-95, Snap25, Bassoon and beta-actin fractions isolated during synaptosomal preparation. Lane 1, nuclear fraction (P1); lane 2, first cytosolic fraction (S1); lane 3, second cytosolic fraction (S2); lane 4, crude synaptosome (P2), lane 5, total brain extracts (see Figure S1 for details). (B) Statistically enriched proteins in the p140Cap IP. The Volcano plot represents the log₁₀(p-value, y axis) plotted against the log₂(fold change, x axis) for proteins quantified in p140Cap IPs from WT and p140Cap KO, used as negative control. 357 different proteins, including p140Cap (arrowhead) were found significantly enriched in WT samples (FDR ≤ 1%, ≥ 4-fold enrichment) are shown as green dots (C) Validation of synaptic p140Cap interacting proteins identified in the interactome. Lane 1 and 5, p140Cap IP in WT animals; lane 2 and 6, p140Cap IP in p140Cap KO animals; lane 3 and 7, input from WT synaptosomes; lane 4 and 8, input from KO synaptosomes. Co-immunoprecipitated proteins are shown on the left, along with their rank in MS. (D) Reverse validation for p140Cap interacting proteins Dlg4/PSD95 and Grin2a/NR2A. Lane 1, Dlg4/PSD95 IP in WT animals; lane 2, Dlg4/PSD95 IP in p140Cap KO animals; lane 4, input from WT synaptosomes; lane 5, input from KO synaptosomes; lane 6, Grin2a/NR2A IP in WT animals; lane 7, Grin2a/NR2A IP in p140Cap KO animals; lane 9, input from WT synaptosomes; lane 10, input from KO synaptosomes. Lane 3 and lane 8 are control IP with IgGs in WT animals.