Novel roles of ER stress in repressing neural activity and seizures through Mdm2- and p53-dependent protein translation
Fig 3
Mdm2 expression and nuclear accumulation are induced during the early phase of ER stress.
(A) Representative western blots of Mdm2 and Gapdh after treatments of vehicle (DMSO) or Thapsigargin (Tg, 1 μM) for 1 hour in WT cortical neuron cultures pre-treated with DMSO, cycloheximide (60 μM) or actinomycin (20 μM) for 10 minutes. Quantification of Mdm2 level is on the right (n = 6). (B) Quantitative real-time RT-PCR of Mdm2 mRNA normalized to Actin mRNA from WT cortical neuron cultures treated with vehicle (DMSO) or Tg for 1 hour (n = 4). (C) Representative western blots of phospho (P)- Mdm2, Mdm2, and Gapdh from WT cortical neuron cultures treated with vehicle (DMSO) or Thapsigargin (Tg, 1 μM) for 1 hour (n = 7). (D) Representative western blots of Mdm2, Lamin A/C and HSP-90 after nuclear and cytosolic extraction using WT cortical neuron cultures treated with vehicle (DMSO) or Tg for 1 hour. Lamin A/C and HSP-90 serve as controls for nuclear and cytosolic fractions, respectively. Quantification is done after normalizing Mdm2 to Lamin A/C (n = 5). (E) Immunocytochemistry of WT cortical neuron cultures treated with vehicle (DMSO) or Thapsigargin (Tg, 1 μM) for 1 hour. Representative Mdm2, DAPI and merged images, as well as quantification of nuclear Mdm2, are shown (n = 20 cells from two independent cultures). Scale bar: 20 μm. For the quantification above, a one-way ANOVA with Tukey test (A) and Student’s t-test (B-E) were used. Data are represented as mean ± SEM with *P<0.05, **P<0.01, ***P<0.001, ns: non-significant.