Fig. 4: Regulation by MTFL₄₅₇ of the TrkB-FL/Hrs interaction induced by excitotoxicity.

A Cortical neurons were preincubated with MTMyc and MTFL₄₅₇ (25 μM, 30 min) and treated with NMDA for the indicated times. Cells were analyzed by immunofluorescence with antibodies for Hrs (green) and TrkB-FL Ct (red), together with DAPI staining (blue). Representative confocal microscopy images correspond to single sections and show the fused channels. Scale bar: 10 μm. B Mean values ± SD of Pearson correlation coefficient (PCC; n = 3). For each independent experiment, a minimum of 80 different neurons were analyzed. Statistical analysis was performed using a generalized linear model followed by a post-hoc Fisher’s LSD test (P = 0.06, 0 vs. 60 min of NMDA treatment in MTMyc-cultures). C, D Analysis by immunoprecipitation of TrkB-FL/Hrs interaction. Cultures preincubated with cell-penetrating peptides (CPPs) as above were treated with NMDA for 30 min and compared to untreated cultures. Immunoprecipitation (IP) was performed with the Hrs antibody, and the immunoprecipitated proteins were analyzed by immunoblot using the same antibody (C) or TrkB-FL Ct (D). Total protein lysates and immunoprecipitated proteins were analyzed in parallel. Mean values ± SD (n = 4) of Hrs and TrkB-FL levels relative to those found in cells preincubated with MTMyc and without NMDA are represented. Statistical analysis was performed using two-way ANOVA followed by a Bonferroni test (*P < 0.05, **P < 0.01).