Anti p35

The Cdk5 kinase activity was measured using a previously established Cdk5 kinase activity assay (Zhang et al., 2010 (link), 2015 (link); Qu et al., 2011 (link); Sheng et al., 2016 (link)). Briefly, mouse hippocampi were homogenized and lysed, and then Cdk5/p35/p25 protein complex was co-immunoprecipitated using Cdk5 antibody (C8, 1 ug/500 g protein lysate) overnight and pulled down by protein-A/G agarose beads at 4°C for 60 min. The beads were then washed three times with lysis buffer and once with kinase reaction buffer. The kinase assay was then performed with 10 μg histone H1 and magnesium/ATP mixture at 30°C for 30 min, followed by western blotting using the phospho-histone H1 antibody. Cdk5, p35, phosphor-tau (p-S202), phosphor-tau (p-S404), tau, and β-actin were blotted using anti-Cdk5 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p35 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-tau (1:2,000; Cell Signaling Technology, Danvers, MA, USA), anti-tau (1:2,000; Cell Signaling Technology, Danvers, MA, USA), and anti-actin (1:5,000; Cell Signaling Technology, Danvers, MA, USA). The band intensity was quantified by chemiluminescence and densitometric scanning of the films under linear exposure conditions using ImageJ software (NIH).