Fig. 3: Effect of excitotoxicity on TrkB-FL interaction with endosomal protein Hrs.

A–C Analysis by immunofluorescence of TrkB-FL/Hrs colocalization. A Cortical neurons were treated with NMDA for the indicated times and analyzed with antibodies specific for Hrs (green) and TrkB-FL Ct (red); nuclear staining was performed with DAPI (blue). Representative images obtained by confocal microscopy correspond to single sections and show the fused channels. Scale bar: 20 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 < 0.05, compared to untreated cultures). C Percentage of cells showing at different times of NMDA treatment a PCC ≥ 0.50, generally considered as a threshold for protein colocalization. Statistical analysis was performed as before (* P < 0.05, compared to untreated cultures; n = 3). D, E Analysis by immunoprecipitation of TrkB-FL/Hrs interaction. Neuronal cultures were treated with NMDA (100 μM) or BDNF (100 ng/ml) for 30 min and compared to untreated cultures. Immunoprecipitation was performed with the Hrs antibody, and the immunoprecipitated proteins (IP) were analyzed by immunoblot using the same antibody (D) or TrkB-FL Ct (E). Total protein lysates were analyzed in parallel with the immunoprecipitated proteins. Mean values ± SD (n = 5, except for BDNF-treated cells where n = 4) of Hrs and TrkB-FL levels in NMDA- or BDNF-treated cultures relative to untreated cells are represented for both total lysates and Hrs-immunoprecipitated proteins. Statistical analysis was performed as above (*P < 0.05, compared to the respective untreated cultures; ^#P < 0.05 and ^##P < 0.01, as indicated).