Fig. 7.

Accumulation of ubiquitinated proteins in hippocampal neurons from fragile X mice. (A) Primary cultures of hippocampal neurons (DIV 14) from WT and Fmr1-KO mice were immunolabeled with antibodies against ubiquitin, p62, and the neuronal marker MAP-2. (Scale bars, 10 µm.) (B and C) Bar graphs show density of ubiquitin puncta per soma (B) and density of puncta colabeled for p62 and ubiquitin per soma (C), each normalized to the corresponding WT value. (D and E) Protein in whole-cell lysates of primary hippocampal neurons from WT and Fmr1-KO mice was immunoprecipitated with an antibody to ubiquitin. Whole-cell lysates (Input) and immunoprecipitates (IP) were probed for PSD-95 (D) and Arc (E). (Top) Representative blots. (Middle) Summary data for input. (Bottom) Summary data for precipitate. All summary data were normalized to the corresponding values for vehicle-treated neurons. *P < 0.05, **P < 0.01. Statistics were calculated by a two-tailed Student’s t test. n = 4 independent experiments from different batches of neurons; a minimum of 50 neurons per batch for each treatment group were analyzed in B and C. Values reflect mean ± SEM.