Fig. 5

Exo70 proximity to the plasma membrane decreases after mTBI induction. a Brains from two-month-old mice were cut in 300 µm thick slices. Biotinylation was carried out and the hippocampus dissected. Hippocampal slices were homogenized in a modified RIPA buffer (see methods). 500 µg of protein samples were incubated with NeutrAvidin overnight at 4 °C and beads were obtained by brief centrifugation. 200 µg (40%) of total protein samples were loaded alongside cytosolic (unbound) and eluted samples from NeutrAvidin precipitation assays. Samples were resolved in 10% SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with the antibodies indicated in the figure. Membranes were stripped and tested again with the indicated antibodies. b Exo70 proximity to the plasma membrane was described as follows: Combined cytosolic and surface Exo70 band intensities were considered as 100% Then Exo70 proximity was calculated using the band intensity combination. The graph shows that nearly 30% of the Exo70 total pool is proximal to the plasma membrane. Mean ± SEM is shown, n = 3 hippocampi from different mice. c Two-month-old male mice were subjected to mTBI and hippocampal slices were prepared and biotinylated. 500 µg of protein samples were incubated with NeutrAvidin overnight at 4 °C and beads were obtained by brief centrifugation. Lysates were analyzed using 30 µg of proteins. d Exo70 densitometric analysis. Protein lysates were used to normalize band intensities. 4 Hippocampal slices from each mouse were used. Values represent means ± SEM, n = 3 mice per experimental group. Statistical differences were determined by an unpaired t-test comparing Sham and mTBI. *  < 0.05