anti yap1  (Novus Biologicals)

Journal: Nature

Article Title: Intestinal macrophages modulate synucleinopathy along the gut–brain axis

doi: 10.1038/s41586-025-09984-y

Figure Lengend Snippet: a , Schematic of gut cross-section. b , Confocal image of duodenal myenteric plexus of 3-month WT versus 3KL. c , Quantification of s129p + αS pathology: n = 6–8 mice; 1 datapoint, average of 1 mouse; 3–10 ROIs per mouse. Three experiments, data analysed using an unpaired t -test. d , Whole gastrointestinal transit time in 3-month WT versus 3KL, n = 9–10. Two experiments, unpaired t -test. e , Hierarchically clustered heatmap of ROI-specific nCounter digital counts across PD-relevant protein targets in duodenal myenteric plexus of 3-month WT versus 3KL, n = 2, 2–3 ROIs per mouse. f – j , Confocal images of engulfed αS by murine MHCII + duodenal ( f ) and human CD209 + jejunal ( g ) ME-Macs, quantified murine LAMP1 lysosomal volume ( h ) and engulfment of s129p in murine ( i ) and αS (2F12) in human ( j ) LAMP1 + lysosomes. n = 8 mice per genotype ( h , i ) and n = 6 NHC and 8 PD postmortem samples ( j ). Two experiments, Mann–Whitney test ( h – j ). k , Volcano plot showing differentially expressed proteins in sorted ME-Macs of 3-month WT versus 3KL. n = 2 per genotype, with 3 mice pooled per biological unit. l , Biological processes enriched in 3-month 3KL ME-Macs, one-sided hypergeometric test. m , Schematic of SAA on sorted duodenal ME-Macs versus enteric neurons. n , o , αS aggregation kinetics through SAA in duodenal enteric neurons ( n ) and ME-Macs ( o ). p , SAA-positive versus negative count in different cell lysates. A sample was counted positive if aggregation onset (lag time) was at least 2 h shorter than negative control (PBS). n = 11 (enteric neurons), n = 12 (ME-Macs). Six experiments, Fisher’s exact test. Data are mean ± s.e.m. (error bars). FC, fold change; GI, gastrointestinal; NO, nitric oxide; ROS, reactive oxygen species; ThT, thioflavin T. Scale bars, 50 μm ( a ); 10 μm ( b ); 5 μm ( f – j ), insets 2 μm ( f – j ).

Article Snippet: Samples were processed and analysed as described before using 2F12 (MABN1817, Merck) as a capture, SOY1 (Merck, MABN1818) as a sulfo-tagged detection antibody on an MSD enzyme-linked immunosorbent assay (ELISA) platform .

Techniques: MANN-WHITNEY, Negative Control

Journal: The Febs Journal

Article Title: The complete absence of cytoplasmic γ‐actin results in no discernible phenotype in mice or primary fibroblasts

doi: 10.1111/febs.70075

Figure Lengend Snippet: Expression of select Actin‐associated proteins is unchanged in Actg1 c‐b/c‐b mouse embryonic fibroblasts (MEFs). Relative protein expression of Actin‐associated proteins SRF, MRTF‐A, and YAP normalized to GAPDH and relative to wild type (WT) in WT, Actg1 c‐b/+ , and Actg1 c‐b/c‐b MEFs ( n = 4). Two‐way ANOVA with Bonferroni posttest was performed. * P < 0.05. Error bars are SEM.

Article Snippet: The following antibodies were used: β‐actin (1:5000; Sigma‐Aldrich, AC15, Burlington, MA, USA), γ‐actin (1:5000 mAB 2–4), α sm ‐actin (1:5000; Sigma‐Aldrich, 1A4, Burlington, MA, USA), Pan‐actin (1:5000; Seven Hills Bioreagents, C4, Cincinnati, OH, USA), SRF (1:1000; Santa Cruz Biotechnology, G‐20, Dallas, TX, USA), MRTF‐A (1:1000; Cell Signaling Technology, E2V2I, Danvers, MA, USA), or YAP (1:500, Abnova, 2F12) with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1:5000; Sigma‐Aldrich, G9545, Burlington, MA, USA) as a loading control and secondary antibodies DyLight 800 anti‐mouse IgG (1:10 000; Cell Signaling Technology, 5257S, Danvers, MA, USA) and DyLight 680 anti‐rabbit IgG (1:10 000; Cell Signaling Technology, 5366S, Danvers, MA, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects

doi: 10.1038/s41419-024-06927-9

Figure Lengend Snippet: Primers used for RT-qPCR.

Article Snippet: For the epitope mapping, peptides were dissolved into a small amount of dimethyl sulfoxide and subsequently diluted into PBS at a concentration of 1 mg/mL, with 5 μg of each peptide dot-blotted onto nitrocellulose membrane and air-dried for 30 min. After blocking the membrane for 1 h with 5% milk in 0.1% Tween-20 in tris-buffered saline (TBS-T), the blots were incubated overnight in a cold room with sdAbs 1D9, 2B8, 1F12, and 2F12 for tau (containing his tag, 0.01 mg/mL) in Superblock (Thermo Fisher Scientific).

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects

doi: 10.1038/s41419-024-06927-9

Figure Lengend Snippet: A The effect of expressing the indicated sdAbs alone (without tauR406W) on the adult lifespan. The survival curves are similar between elav-GAL4/Y controls and the different sdAbs with no significant difference. Genotypes and the number of flies analyzed per group: 1. elav-GAL4/Y ( n = 86), 2. elav-GAL4/Y; UAS-1D9/+ ( n = 109), 3. elav-GAL4/Y; UAS-2B8/+ ( n = 53), 4. elav-GAL4/Y; UAS-1F12/+ ( n = 64), and 5. elav-GAL4/Y; UAS-2F12/+ ( n = 102). B The survival curves are very significantly different between elav-GAL4/Y controls and tauR406W flies ( p < 0.0001) with control flies surviving the longest and tauR406W flies surviving the shortest. Both the EGFP and Dv VHH control lines exhibited a shorter lifespan. The sdAb 2F12 did not extend the lifespan ( p = 0.4110) when compared to the negative controls EGFP and Dv VHH , whereas sdAbs 1D9 and 1F12 extended the lifespan by 20 days (Log-rank test, p < 0.0001). The anti-tau sdAb 2B8 co-expressed with tauR406W flies live as long as the elav-GAL4 controls ( p < 0.0001). Genotypes and the number of flies analyzed per group: 1. elav-GAL4/Y ( n = 300), 2. elav-GAL4/Y;; UAS-tauR406W/+ ( n = 170), 3. elav-GAL4/Y; UAS-EGFP/ + ; UAS-tauR406W/+ ( n = 275), 4. elav-GAL4/Y; UAS-Dv VHH /+; UAS-tauR406W/+ ( n = 250), 5. elav-GAL4/Y; UAS-1D9/ + ; UAS-tauR406W/+ ( n = 200), 6. elav-GAL4/Y; UAS-2B8/ + ; UAS-tauR406W/+ ( n = 300), 7. elav-GAL4/Y; UAS-1F12/ + ; UAS-tauR406W ( n = 200), and 8. elav-GAL4/Y; UAS-2F12/ + ; UAS-tauR406W ( n = 200).

Article Snippet: For the epitope mapping, peptides were dissolved into a small amount of dimethyl sulfoxide and subsequently diluted into PBS at a concentration of 1 mg/mL, with 5 μg of each peptide dot-blotted onto nitrocellulose membrane and air-dried for 30 min. After blocking the membrane for 1 h with 5% milk in 0.1% Tween-20 in tris-buffered saline (TBS-T), the blots were incubated overnight in a cold room with sdAbs 1D9, 2B8, 1F12, and 2F12 for tau (containing his tag, 0.01 mg/mL) in Superblock (Thermo Fisher Scientific).

Techniques: Expressing, Control

Journal: Cell Death & Disease

Article Title: Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects

doi: 10.1038/s41419-024-06927-9

Figure Lengend Snippet: A Domain organization of the largest isoform of tau (2N4R) with 441 amino acids showing peptides 16 and 18. B Quantification of a dot blot assay on the binding of sdAb 2B8 to peptides covering all of the 441 amino acids of the tau protein. Each peptide within the library was 25 amino acids except peptide 25, which has 9 amino acids. Each peptide had a 7 amino acid overlap with the following peptide. Only peptides 16 (tau 271–295) and 18 (tau 307–331) displayed strong reactivity with 2B8. Positive control: PHF-enriched tau protein from human tauopathy brain. Negative control: BSA. Each bar shows the mean normalized signal ± SD of three replicates. C 2B8 sdAb bound to peptide 16 in solution phase BLI assay with high affinity ( K D = 12.1 ± 6.6 nM). D 2B8 sdAb bound to peptide 18 in the solution phase with high affinity ( K D = 5.2 ± 1.7 nM). The sdAb is attached to the biosensor, which reacts with different concentrations of peptide 16 and peptide 18. The resulting curves display the shift in wavelength interference in nanometers (nm), which is represented as binding. The curves depict the association and dissociation of sdAbs and tau peptides at different concentrations of tau peptides. The broken line represents the fitting curve used to calculate the K D value ± SD which is calculated from three independent experiments. E Quantification of a dot blot assay on the binding of sdAb 1D9 to tau peptides showed strong binding to peptide 16 and moderate binding to peptide 18. F The sdAb 1D9 bound to peptide 16 with high affinity in the solution phase ( K D = 13.4 ± 3.6 nM). G The binding affinity of sdAb 1D9 to peptide 18 in the solution phase was rather high ( K D = 23.6 ± 0.5 nM). H Quantification of a dot blot assay on the binding of sdAb 1F12 to tau peptides showed strong binding to peptide 18 and moderate binding to peptide 16. I The binding affinity of sdAb 1F12 to peptide 16 in the solution phase was moderate ( K D = 1.26 ± 0.7 μM). J The sdAb 1F12 did not bind to peptide 18 in the solution phase. K Quantification of a dot blot assay on the binding of sdAb 2F12 to tau peptides showed strong binding to peptide 16 and moderate binding to peptide 18. L The sdAb 2F12 did not bind to peptide 16 in the solution phase. M 2F12 sdAb bound to peptide 18 in the solution phase with a high affinity ( K D = 2.98 ± 0.2 nM). See Supplemental Fig. for dot blot assay images and Table for the binding affinity of all sdAbs. This data for 2B8 has been previously reported .

Article Snippet: For the epitope mapping, peptides were dissolved into a small amount of dimethyl sulfoxide and subsequently diluted into PBS at a concentration of 1 mg/mL, with 5 μg of each peptide dot-blotted onto nitrocellulose membrane and air-dried for 30 min. After blocking the membrane for 1 h with 5% milk in 0.1% Tween-20 in tris-buffered saline (TBS-T), the blots were incubated overnight in a cold room with sdAbs 1D9, 2B8, 1F12, and 2F12 for tau (containing his tag, 0.01 mg/mL) in Superblock (Thermo Fisher Scientific).

Techniques: Dot Blot, Binding Assay, Positive Control, Negative Control

Journal: Cell Death & Disease

Article Title: Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects

doi: 10.1038/s41419-024-06927-9

Figure Lengend Snippet: Binding affinities of anti-tau sdAbs to peptide 16 and peptide 18 of the 2N4R isoform of recombinant tau comprising of 441 amino acids.

Article Snippet: For the epitope mapping, peptides were dissolved into a small amount of dimethyl sulfoxide and subsequently diluted into PBS at a concentration of 1 mg/mL, with 5 μg of each peptide dot-blotted onto nitrocellulose membrane and air-dried for 30 min. After blocking the membrane for 1 h with 5% milk in 0.1% Tween-20 in tris-buffered saline (TBS-T), the blots were incubated overnight in a cold room with sdAbs 1D9, 2B8, 1F12, and 2F12 for tau (containing his tag, 0.01 mg/mL) in Superblock (Thermo Fisher Scientific).

Techniques: Binding Assay, Recombinant

Journal: Cell Death & Disease

Article Title: Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects

doi: 10.1038/s41419-024-06927-9

Figure Lengend Snippet: Light microscopy images of 5-day-old adult eyes. A A control fly eye not expressing tauR406W (genotype, elav-GAL4/Y ) shows a standard pattern of ommatidia and mechanosensory bristles. B – D The retinal organization is disrupted in tauR406W-expressing control fly eyes, with ommatidial fusions and missing bristles. Genotypes: B elav-GAL4/Y; UAS-tauR406W/+ . C elav-GAL4/Y; UAS-EGFP/ + ; UAS-tauR406W/+ . D elav-GAL4/Y; UAS-Dv VHH /+; UAS-tauR406W/+ . E Expressing anti-tau sdAb 1D9 did not rescue the tauR406W-induced phenotype. Genotype, elav-GAL4/Y; UAS-1D9/ + ; UAS-tauR406W/+ . F However, the ommatidial fusion phenotype was rescued by expressing 2B8. Genotype: elav-GAL4/Y; UAS-2B8/ + ; UAS-tauR406W/+ . About 5% of the 2B8 flies ( n = 100) showed dark spots indicated by blue arrow ( G ). Expressing 1F12 ( H , genotype: elav-GAL4/Y; UAS-1F12/ + ; UAS-tauR406W ) and 2F12 ( I , genotype: elav-GAL4/Y; UAS-2F12/ + ; UAS-tauR406W ) did not suppress the tauR406W phenotype. The semi-quantitative score of the eye images for each genotype is represented in J (Kruskal–Wallis, overall p < 0.0001, Dunn’s post-hoc test, ** p ≤ 0.01). K – N Representative scanning electron microscope (SEM) images of 5-day-old adult fly eyes of the indicated genotypes: K elav-GAL4/Y , L elav-GAL4/Y;; UAS-tauR406W/+ , M elav-GAL4/Y; UAS-1D9/ + ; UAS-tauR406W/+ , and N elav-GAL4/Y; UAS-2B8/ + ; UAS-tauR406W/+ . The scale bar represents 100 μm.

Article Snippet: For the epitope mapping, peptides were dissolved into a small amount of dimethyl sulfoxide and subsequently diluted into PBS at a concentration of 1 mg/mL, with 5 μg of each peptide dot-blotted onto nitrocellulose membrane and air-dried for 30 min. After blocking the membrane for 1 h with 5% milk in 0.1% Tween-20 in tris-buffered saline (TBS-T), the blots were incubated overnight in a cold room with sdAbs 1D9, 2B8, 1F12, and 2F12 for tau (containing his tag, 0.01 mg/mL) in Superblock (Thermo Fisher Scientific).

Techniques: Light Microscopy, Control, Expressing, Microscopy