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. 2018 Apr 30;37(11):e97943. doi: 10.15252/embj.201797943

Figure EV4. NMDAR‐dependent decrease in APT1 expression is insensitive to Akt inhibition or Ago2 phosphorylation at S387 (related to Fig 7).

Figure EV4

  1. Endogenous APT1 protein levels are rapidly reduced in response to NMDAR stimulation. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, and lysates were prepared 10, 20 or 40 min after NMDA washout and analysed by Western blotting. Graphs show quantification of APT1 expression normalised to vehicle control; n = 6. **P < 0.01; ***P < 0.001 one‐way ANOVA, Bonferroni post hoc test. Error bars are SEM.
  2. NMDAR‐dependent decrease in APT1 is Akt‐independent. Cortical neuronal cultures were treated with Akti‐1/2 20 min before NMDA or vehicle application, and lysates were prepared 40 min after NMDA washout and analysed by Western blotting. Graphs show quantification of APT1 expression normalised to vehicle control; n = 6. *P < 0.05; two‐way ANOVA, Bonferroni post hoc test. Error bars are SEM.
  3. NMDAR‐dependent decrease in dendritic APT1 expression is unaffected by Ago2 phosphorylation at S387. Cortical neurons were transfected with molecular replacement constructs expressing Ago2 shRNA plus shRNA‐resistant GFP‐Ago2 (WT, S387A or S387D), fixed 40 min after NMDA washout, permeabilised and stained with APT1 antibodies (red channel). GFP signal was maximised at acquisition so that dendrites could be effectively visualised. Graph shows APT1 staining intensity in dendrites normalised to vehicle control. Scale bar = 20 μm; n = 10 cells from five independent experiments. ***P < 0.001 two‐way ANOVA, Bonferroni post hoc test. Error bars are SEM.

Source data are available online for this figure.