Figure 4

Regulation of BMAL1 protein stability by Aβ-mediated sumoylation. (A & B) Aβ affected the stability of BMAL1 protein. HT22 cells were incubated with CHX (100 μg/ml) with vehicle, or CHX (100 μg/ml) with Aβ (2 μM) for the indicated times. The protein level of BMAL1 was analyzed by immunoblotting with anti-BMAL1 antibody and then quantified by densitometry. Data are represented as mean ± SEM. ^** P < 0.01, ^*** P < 0.001. (C & D) Effects of Aβ on the interaction between BMAL1 and SUMO1. (C) HT22 cells were incubated with vehicle or Aβ (2 μM) for 24 h after cell synchronization. (D) Both littermate and 5XFAD mice brains were prepared for immunoprecipitation. Each sample was immunoprecipitated using the anti-BMAL1 antibody. Confirmation by western blot analysis with anti-SUMO-1 antibody showed that the immunoprecipitated BMAL1 was physically interacted with SUMO1. This interaction was greater with Aβ treatment. (E) The transfection of Sumo1 siRNA into HT22 cells rescued Aβ-induced BMAL1 degradation compared to control siRNA. (F) Densitometric analysis of BMAL1 protein from three independent experiments. BMAL1 was normalized to α-Tubulin. ^** P < 0.01, ^# P < 0.05, ^$ P < 0.05. (G) The transfection of BMAL1 K259R into HT22 cells decreased Aβ-induced BMAL1 degradation compared to BMAL1 WT. (H) Densitometric analysis of BMAL1 protein from three independent experiments. BMAL1 was normalized to GAPDH. ^*** P < 0.001, ^# P < 0.05.