Western Blot Analysis of CDK5 and p35 in Hippocampus

After the treatments, the neuron cultures or the medium lateral hippocampus, including the CA1 and dentate gyrus (DG), were homogenised in lysis buffer (Cytoskeleton, Biochem), and Western blotting was performed as previously described (Piedrahita et al. 2010 (link)). The membranes were incubated overnight at 4°C with the following primary antibodies: rabbit p35 1:250 (C-19, Santa Cruz), rabbit CDK5 1:250 (Santa Cruz), and mouse AT8 1:500 (Pierce). βIII tubulin 1:5000 (Promega) was used as a loading control. IRDye 800CW goat anti-mouse or IRDye 680 goat anti-rabbit 1:15000 (LI-COR) were used as secondary probes. The blots and images were developed and analysed using Odyssey Infrared Imaging System application software version 3.0 (LI-COR). The quantification of each blot is presented as the relative units with respect to the loading control values.

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Posada-Duque R.A., López-Tobón A., Piedrahita D., González-Billault C, & Cardona-Gomez G.P. (2015). p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing. Journal of neurochemistry, 134(2), 354-370.

Publication 2015

βIII tubulin IRDye 800CW goat anti-mouse IRDye 680 goat anti-rabbit Odyssey Infrared Imaging System application software version 3.0

Antibodies Buffer Cdk5 Cytoskeleton Dentate gyrus Goat Hippocampus Irdye 800cw Membranes Mouse Neuron Promega Rabbit Tubulin

Corresponding Organization :

Other organizations : Universidad de Antioquia, University of Chile

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Variable analysis

independent variables

Treatments (not explicitly specified)

dependent variables

Levels of p35
Levels of CDK5
Levels of phosphorylated tau (AT8)

control variables

βIII tubulin (used as a loading control)

controls

No positive or negative controls were explicitly mentioned.

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