Figure 9. Cdk5 is implicated in the neuronal activity–dependent promotion of β cleavage and microdomain switching. (a) Effects of neuronal hyperactivity on β cleavage of endogenous APP. At 11–12 DIV, cortical neurons were treated with PTX and a cdk5 inhibitor in the presence of a γ-secretase inhibitor for 48 h. Accumulated CTFs generated from endogenous APP by α or β cleavage were quantified with anti-APPC15. PTX treatment caused a twofold increase in β cleavage, which was reduced significantly by cdk5 inhibitors. Results are means + SD of 10 measurements. (b) Effects of KCl treatment on microdomain switching. At 3 DIV, hippocampal neurons were transfected with APP, syntaxin 1–HA, and BACE1AA. At 6 DIV, neurons were treated with 25 mM KCl and roscovitine for 20 h. Switching of microdomain association was induced by KCl and substantially inhibited by roscovitine. Data are means + SD based on three independent experiments (n = 33–36). (c) A PTX-induced shift of APP association to BACE1-containing DRMs. At 11 DIV, cortical neurons were treated with PTX and olomoucine for 48 h. Before DRM preparation, neurons were treated with the membrane-permeable BACE1 inhibitor for 5 h. DRMs were prepared and used for IP with anti-BACE1c. PTX treatment increased the association of endogenous APP with BACE1-containing DRMs, which was reduced by olomoucine treatment. (d) High K+-induced promotion of copatching of endogenous APP and BACE1 in neurons. At 7–8 DIV, hippocampal neurons were treated with 25 mM KCl and roscovitine for 24 h. Patching of endogenous APP and endogenous BACE1 was induced with rabbit anti-APPex and chicken anti-BACE1ex. A KCl-induced increase in endogenous APP–BACE1 copatching and partial inhibition by roscovitine validated the copatching analysis of exogenously expressed proteins. Data are means + SD based on three independent experiments (n = 31 or 32). *, P < 0.05; **, P < 0.01.
Figure 9.

Cdk5 is implicated in the neuronal activity–dependent promotion of β cleavage and microdomain switching. (a) Effects of neuronal hyperactivity on β cleavage of endogenous APP. At 11–12 DIV, cortical neurons were treated with PTX and a cdk5 inhibitor in the presence of a γ-secretase inhibitor for 48 h. Accumulated CTFs generated from endogenous APP by α or β cleavage were quantified with anti-APPC15. PTX treatment caused a twofold increase in β cleavage, which was reduced significantly by cdk5 inhibitors. Results are means + SD of 10 measurements. (b) Effects of KCl treatment on microdomain switching. At 3 DIV, hippocampal neurons were transfected with APP, syntaxin 1–HA, and BACE1AA. At 6 DIV, neurons were treated with 25 mM KCl and roscovitine for 20 h. Switching of microdomain association was induced by KCl and substantially inhibited by roscovitine. Data are means + SD based on three independent experiments (n = 33–36). (c) A PTX-induced shift of APP association to BACE1-containing DRMs. At 11 DIV, cortical neurons were treated with PTX and olomoucine for 48 h. Before DRM preparation, neurons were treated with the membrane-permeable BACE1 inhibitor for 5 h. DRMs were prepared and used for IP with anti-BACE1c. PTX treatment increased the association of endogenous APP with BACE1-containing DRMs, which was reduced by olomoucine treatment. (d) High K+-induced promotion of copatching of endogenous APP and BACE1 in neurons. At 7–8 DIV, hippocampal neurons were treated with 25 mM KCl and roscovitine for 24 h. Patching of endogenous APP and endogenous BACE1 was induced with rabbit anti-APPex and chicken anti-BACE1ex. A KCl-induced increase in endogenous APP–BACE1 copatching and partial inhibition by roscovitine validated the copatching analysis of exogenously expressed proteins. Data are means + SD based on three independent experiments (n = 31 or 32). *, P < 0.05; **, P < 0.01.

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