Fig 2: Expression levels of membrane-bound CB1R and AMPAR subunit GluA2 in females and males. (A) Representative Western blots of membrane-bound CB1R and GluA2, in females and (D) males. (B) Quantification of membrane-bound CB1R levels in females (C, n = 5; PTZ, n = 8; WIN, n = 5; WIN + PTZ, n = 6). (C) Quantification of membrane-bound GluA2 levels in females (C, n = 5; PTZ, n = 8; WIN, n = 5; WIN + PTZ, n = 7). (E) Quantification of membrane-bound CB1R levels in males (C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5). (F) Quantification of membrane-bound GluA2 levels in males (C, n = 4; PTZ, n = 6; WIN, n = 4; WIN + PTZ, n = 5). N-cadherin was used as protein loading control for all gels.

Fig 3: Expression levels of proteins obtained from a fraction enriched in cytoplasmic proteins/proteins bound to small organelles. (A) Representative Western blots of CB1R, GluA2R, βArr2, PSD95, p-CaMKII and total CaMKII in females and (I) males. (B–H) Quantification of (A) (Females. CB1R: C, n = 5; PTZ, n = 8; WIN, n = 4; WIN + PTZ, n = 6; GluA2: C, n = 5; PTZ, n = 9; WIN, n = 5; WIN + PTZ, n = 6; βArr2: C, n = 5; PTZ, n = 9; WIN, n = 5; WIN + PTZ, n = 6; PSD95: C, n = 5; PTZ, n = 8; WIN, n = 5; WIN + PTZ, n = 6, p-CaMKIIα: C, n = 5; PTZ, n = 8; WIN, n = 4; WIN + PTZ, n = 5; p-CaMKIIβ: C, n = 5; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5; total CaMKII: C, n = 3; PTZ, n = 4; WIN, n = 3; WIN + PTZ, n = 3). (J–P) Quantification of (D) (Males. CB1R: C, n = 4; PTZ, n = 6; WIN, n = 3; WIN + PTZ, n = 5; GluA2: C, n = 4; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5; βArr2: C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5; PSD95: C, n = 4; PTZ, n = 6; WIN, n = 4; WIN + PTZ, n = 5, p-CaMKIIα: C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5; p-CaMKIIβ: C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5; total CaMKII: C, n = 3; PTZ, n = 4; WIN, n = 3; WIN + PTZ, n = 3). Tubulin was used as protein loading control for all gels. *p < 0.05 and **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons post hoc.

Fig 4: Assessment of surface expression of AMPA receptor GluA2 in hippocampus by immunocytochemistry. (A) Representative images of immunohistochemistry of total (tGluA2) and surface (sGluA2) AMPA receptor GluA2 in wildtype and A350V IQSEC2 mutant hippocampi. Higher power representative images of the CA1 and CA3 regions are shown (right panel). Labeled scale bar for high-resolution images = 20 µm. (B) Quantification of the normalized total GluA2 levels. Data represent mean ± SEM, n = 5 male mice in each group. No significant difference (n.s) P > 0.05, t-test, two-tailed. (C) Quantification of the normalized levels of surface GluA2 expression. Data represent mean ± SEM, n = 5 male mice in each group, *p < 0.05, n.s. p > 0.05, t-test, two-tailed.

Fig 5: mTBI increases exocyst complex assembly and interaction between Exo70 and GluN2B in the PSD fraction. Two-month-old male mice were subjected to mTBI and hippocampal PSD/nonPSD fractions were obtained. 50 and 300 µg of PSD (a) and nonPSD (c) protein samples, respectively, were immunoprecipitated using a specific Exo70 polyclonal antibody. Beads were recovered by brief centrifugation. Samples were resolved in 6% SDS-PAGE and transferred to PVDF membranes. Signal intensity was normalized with Exo70 band intensity. Both hippocampi of each mouse were used. Membranes were stripped and tested again with the indicated antibodies. b, d GluN2A, GluN2B, GluR1, GluR2, Sec6, and Sec10 densitometric analysis from PSD and nonPSD immunoprecipitations. The analysis shows the assembly level of the exocyst complex and Exo70-GluN2B increased interaction after mTBI. Values represent means ± SEM, n = 3 mice per experimental group. Statistical differences were determined by an unpaired t-test comparing Sham and mTBI. *p < 0.05