Anti tau

Anti-APP antibody (6E10) was purchased from Covance (Cat# SIG-39320-1000). Anti-beta amyloid was purchased from Abcam (Cat#2539). Anti-T. gondii antibody was purchased from US biologicals (Cat# T8075-01). BAG1 was a gift from Dr. Dubey. Normal mouse IgG (SC-2025) and normal rabbit IgG (SC-2027) were used as isotype controls to test the specificity of the 6E10 and BAG1 antibodies, respectively. Anti-phospho-Tau antibody (AT8) was purchased from Thermofisher (Cat# MN1020). Anti-NeuN was purchased from Cell Signaling (Cat# 12943S) or Abcam (ab104224). Anti-VGLUT2 (Cat#71555S), anti-GAPDH (Cat# 2118), and anti-Tau (Cat#4019) antibodies were purchased from Cell Signaling. Anti-NMDAR (Cat# ab17345), anti-VGLUT1 (Cat# ab134283), and anti-GAD67 (Cat# ab26116) antibodies were purchased from Abcam. Thioflavin S was purchased from (Sigma Aldrich) and neuro-tracer from Life Technologies.

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).

Immunostaining was performed as previously reported^{50 (link),51} . After fixation, permeabilization and blocking, autaptic neurons cultures were incubated with the following primary antibodies: anti-MAP2 (guineapig polyclonal, Synaptic Systems, Goettingen, Germany, Cat. No. 188 004, 1:1000 dilution)^{50 (link)}, anti-VGLUT1 (rabbit polyclonal, Synaptic Systems, Cat. No. 135 302, 1:2000 dilution)^{50 (link)}, anti-VGAT (rabbit polyclonal, Synaptic Systems, Cat. No. 131 002, 1:1000 dilution), and anti-tau (mouse monoclonal, Cell Signaling Technology, Beverly, MA, USA, Cat. No. 4019, 1:1000 dilution). Then, samples were incubated with the appropriate species-specific fluorochrome-labelled goat secondary antibodies [Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA, Cat. No. A11073) for MAP2, Alexa Fluor 594 (Invitrogen, Cat. No. A11037) for VGLUT1 and VGAT, and CF 488A (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. SAB4600042) for tau, 1:400 dilution]. Samples were mounted in mounting medium containing DAPI (ProLong Gold Antifade Mountant with DAPI, Invitrogen, Cat. No. P36931).
Confocal images of neurons were captured as previously reported⁵¹ . Synaptic puncta were analysed according to previously reported procedures^{50 (link)–52 (link)}. Dendritic and axonal morphologies were analysed using NeuronJ, which was accessed as an ImageJ plugin (v1.4.3, Erik Meijering).