rabbit anti ankle2  (Cell Signaling Technology Inc)

Journal: Life Science Alliance

Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

doi: 10.26508/lsa.202201689

Figure Lengend Snippet: (A) Schematic representation of the workflow for functional validation. Individual sgRNAs were used to silence the corresponding genes in combination with KRAB-dCas9 in tau biosensor cells (BSKRAB-KD), then treated with either sarkosyl-insoluble tau (vesicle-free tau seeds) or exosome-like EVs for 72 h, followed by detection and quantification of tau aggregation using FRET flow cytometry. A fraction of the same BSKRAB-KD cells was also grown for 72 h to corroborate the knockdown of protein expression using Western blots. (B, C, D, E, F, G) Integrated FRET intensities represent levels of tau aggregation upon knocking down the different targets. The control black column (NT) is the average obtained with three independent non-targeting sgRNAs (n = 3) assessed in triplicate. Control cells were compared with knockdown cells targeted individually (1, 2, and 3). Error bars represent the SEM for n = 3, * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001. Each single targeting sgRNA increased tau aggregation with both exosomal and vesicle-free tau seeds. (C, D) Interestingly, ANKLE2 and BANF1 (C, D) appear to induce a stronger effect on tau aggregation induced by exosome-like EVs. (H) Quantitative Western blot analysis of BSKRAB-KD knockdown cells. Each sgRNA generated a protein knockdown of the targeted gene. Note that the ANKLE2-specific antibody reacted with several isoforms, including the canonical variant sized 104–117 kD (red box outline); however, all isoforms were down-regulated when the ANKLE2 locus was silenced. Similarly, the EIF1AD antibody recognized the canonical isoform of 19 kD and one additional variant of lower molecular weight, both being silenced with the individual sgRNA against EIF1AD. (I) Quantification of the extent of protein knockdown for the different targeted genes. Error bars represent the SEM for n = 3, * P < 0.05; ** P < 0.01; and **** P < 0.0001.

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

Techniques: Functional Assay, Biomarker Discovery, Flow Cytometry, Knockdown, Expressing, Western Blot, Control, Generated, Variant Assay, Molecular Weight

Journal: Life Science Alliance

Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

doi: 10.26508/lsa.202201689

Figure Lengend Snippet: (A) FACS plots of knockdown and control cells (non-targeting sgRNA) without adding exogenous tau seeds. Quadrants (Q2 shaded in yellow) in which FRET-positive cells were detected revealed the absence of a FRET signal in both control (NonT) and knockdown cells for ANKLE2, BANF1, VPS18, EIF1AD, and NUSAP1, indicating that no spontaneous tau aggregation was initiated. (B) However, these cells (bottom panels) showed FRET-positive cells only after tau seeds (400 ng of sarkosyl tau) were added, implying the requirement for an exogenous tau seed to trigger the aggregation of endogenous tau. Percentages of FRET-positive cells in Q2 are shown (n = 3, average ± SEM, 40,000 cells/experiment were analyzed).

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

Techniques: Knockdown, Control

Journal: Life Science Alliance

Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

doi: 10.26508/lsa.202201689

Figure Lengend Snippet: (A) Western blot analysis of validated novel regulators using postmortem cortical brain samples (superior frontal cortex) 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 Ser422 (pS422 antibody) in the human brain samples. (C, D, E, F) Quantification of the protein levels in control and AD samples revealed that VPS18 (C), NUSAP1 (D), and EIF1AD (E) are significantly down-regulated in AD patients. (F) However, ANKLE2 levels (F) were not statistically different. Of note, the canonical isoform with a size of 104–117 kD (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/hTau-13 revealed a substantial increase in tau phosphorylation at Ser422 in AD samples. Error bars represent the SEM for n = 6, * P < 0.05; ** P < 0.01; and ns, not significant.

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

Techniques: Western Blot, Control, Phospho-proteomics