Journal: bioRxiv

Article Title: Genome-wide CRISPRi screening reveals regulators of Alzheimer’s tau pathology shared between exosomal and vesicle-free tau seeds

doi: 10.1101/2022.04.26.489622

Figure Lengend Snippet: ( a ) Western blot analysis of validated novel regulators using post mortem cortical brain samples from Alzheimer’s patients (AD). For the pathological data of the patients see . Antibodies against human VSP18, NUSAP1, EIF1AD and ANKLE2 were used. ( b ) Detection of human tau (Tau-13 antibody) and tau phosphorylated at Ser-422 (pS422 antibody) in the human brain samples. ( c-f ) Quantification of the protein levels in control and AD samples reveals that VPS18 ( c ), NUSAP1 ( d ) and EIF1AD ( e ) are significantly downregulated in AD patients. However, ANKLE2 levels ( f ) were not statistically different between both types of brain samples analyzed. Of note, the canonical isoform with a size of 104-117 kDa (red box outline) was the predominant isoform detected in human brains, and therefore, only this isoform was quantified. ( g ) Quantification of the ratio of pS422/hTau13 shows a substantial increase in tau phosphorylation at Ser-422 in AD samples.

Article Snippet: The following antibodies were used: Anti-human ANKLE2 rabbit polyclonal (1:1,000; Thermo A302-965A), anti-human EIF1AD rabbit polyclonal (1:1,000; Thermo 20528-1-AP), anti-BANF1/BAF rabbit monoclonal (1:1,000; Abcam ab129184), anti-human NUSAP1 rabbit polyclonal (1:500; Abcam ab137230), anti-human VPS18 rabbit monoclonal (1:1,000; Abcam ab178689), anti-human Tau-13 mouse monoclonal (1:1,000; Covance MMS-520R), anti Tau phospho Ser422 rabbit polyclonal (1:1,000; GeneTex GTX86147), and the normalizers anti-GAPDH mouse monoclonal (1:2,000; Millipore MAB374) and anti-GAPDH rabbit polyclonal (1:2,000; Proteintech 10494-1-AP).

Techniques: Western Blot