Journal: bioRxiv

Article Title: Knockout of TSPO delays and reduces amyloid, Tau, astrocytosis and behavioral dysfunctions in Alzheimer’s disease

doi: 10.1101/2022.03.26.485919

Figure Lengend Snippet: a , Quantification of mRNA and protein in the whole hippocampus. From left to right, mRNA quantification (2 ^ - (ΔΔCT) ), representative immunoblot (showing two samples from 3xTgAD and two samples from 3xTgAD.TSPO −/− mice) and protein quantification (normalized to ACTIN) for GFAP, VIMENTIN (VIM) and CLUSTERIN (CLU) (two-tailed unpaired t-test: p>0.05). b , Representative example of GFAP (red), IBA1 (green) immunoreactivity and DAPI (blue in merge images) in 9-month-old 3xTgAD and 3xTgAD.TSPO −/− mice. Scale bar: 200 μm. c , Quantification of % of positive GFAP-ir area in the subiculum (Sub), dorsal hippocampus (dHipp) and ventral hippocampus (vHipp), and intensity of the GFAP-ir staining the subiculum (%GFAP + , two-way ANOVA, Subiculum: genotype x age, F 2,24 = 3.16, P = 0.06; genotype, F 1,24 = 26.35, P < 0.0001; age, F 2,24 = 3.03, P = 0.06; dHipp: genotype x age, F 2,25 = 1.06, P > 0.05; genotype, F 1,25 = 6.19, P = 0.019; age, F 2,25 = 1.75, P > 0.05; vHipp: genotype x age, F 2,24 = 0.83, P > 0.05; genotype, F 1,24 = 5.07, P = 0.034; age, F 2,24 = 0.86, P > 0.05; GFAP + intensity, two-way ANOVA, Subiculum: genotype x age, F 2,22 = 3.00, P = 0.07; genotype, F 1,22 = 34.95, P < 0.0001; age, F 2,22 = 3.32, P = 0.055). d , Representative example of VIM (red), STAT3 (green) immunoreactivity and DAPI (blue in merge images) in 9-month-old 3xTgAD and 3xTgAD.TSPO −/− mice. Scale bar: 200 μm (inserts display high magnification). e , Positive control for VIM (left), STAT3 (right) immunoreactivity and merge images with DAPI (20-month-old 3xTgAD mouse). Scale bar: 200 μm. f , Quantification of % of positive VIM-ir area in dHipp and vHipp (%VIM + , two-way ANOVA, dHipp: genotype x age, F 2,25 = 0.78, P > 0.05; genotype, F 1,25 = 0.04, P > 0.05; age, F 2,25 = 0.56, P > 0.05; vHipp: genotype x age, F 2,25 = 0.49, P > 0.05; genotype, F 1,25 = 0.92, P > 0.05; age, F 2,25 = 4.36, P = 0.024). g , Representative example of the signal extraction for Sholl analysis (GFAP, red; IBA1, green; DAPI, blue). Image on the right shows a high magnification of the insert. Scale bar: 20 μm. h , Quantification of the number of intersections as function of the distance from soma in dorso-dorsal (ddHipp), dorso-lateral (dlatHipp) hippocampus and hilus (two-way ANOVA, ddHipp: genotype x distance, F 28,224 = 3.02, P < 0.0001; genotype, F 1,8 = 5.34, P = 0.049; distance, F 28,224 = 129.7, P < 0.0001; dlatHipp: genotype x distance, F 28,224 = 0.88, P = 0.63; genotype, F 1,8 = 1.1, P > 0.05; distance, F 28,224 = 132.9, P < 0.0001; hilus: genotype x distance, F 28,224 = 4.15, P < 0.0001; genotype, F 1,8 = 5.96, P = 0.04; distance, F 28,224 = 151.9, P < 0.0001). i , Total number of ramifications (two-way ANOVA: genotype x area, F 2,24 = 0.46, P > 0.05; genotype, F 1,24 = 22.87, P < 0.001; area, F 2,24 = 3.80, P = 0.036). j , Size of the soma of astrocytes (two-way ANOVA: genotype x area, F 2,24 = 0.26, P > 0.05; genotype, F 1,24 = 11.4, P = 0.0025; area, F 2,24 = 8.71, P = 0.0014).

Article Snippet: Primary antibodies used at 1/250 were as follows: Actin (Sigma), ApoE (Abcam), BACE1 (Cell signaling), Clusterin (Santa Cruz), GFAP-Cy3 (Sigma), IDE (Abcam), TSPO (Abcam) and secondary antibodies (1/1000) were Alexa Fluor antibodies from Thermo Fisher.

Techniques: Western Blot, Two Tailed Test, Staining, Positive Control

Journal: Heliyon

Article Title: Identification of novel substrates of a disintegrin and metalloprotease 17 by specific labeling of surface proteins

doi: 10.1016/j.heliyon.2020.e05804

Figure Lengend Snippet: Identification of protein spots by MS.

Article Snippet: The membranes were incubated in a solution containing mouse anti-ADAM17 antibody (sc-390859, Santa Cruz Biotechnology), mouse anti-L1CAM antibody (sc-53386, Santa Cruz Biotechnology), mouse anti-desmoglein-2 antibody (sc-80663, Santa Cruz Biotechnology), rabbit anti-galectin-3 antibody (ab76245, Abcam), rabbit anti-galectin-3 binding protein (G3BP) antibody (ab217572, Abcam), rabbit anti-clusterin antibody (42143S, Cell Signaling Technology, MA, USA), and mouse anti-beta-actin (ACTB) antibody (sc-47778, Santa Cruz Biotechnology).

Techniques: Sequencing

Journal: Heliyon

Article Title: Identification of novel substrates of a disintegrin and metalloprotease 17 by specific labeling of surface proteins

doi: 10.1016/j.heliyon.2020.e05804

Figure Lengend Snippet: Effects of ADAM17 knockdown on the shedding of clusterin and G3BP. Detection of clusterin by 1D- (A) and 2D-western blotting (B). (C) Detection of clusterin in the culture medium. Protein samples (10 μg) were loaded into each well. Western blot (upper). Quantification of the bands (lower). Detection of G3BP (D) and galectin-3 (E) by western blotting. (F) Detection of G3BP in the culture medium. Eluted protein samples (10 μg) were loaded into each well. Western blot (upper). Quantification of the bands (lower). Full-unadjusted images are shown in supplementary content (Supplementary Figure 3).

Article Snippet: The membranes were incubated in a solution containing mouse anti-ADAM17 antibody (sc-390859, Santa Cruz Biotechnology), mouse anti-L1CAM antibody (sc-53386, Santa Cruz Biotechnology), mouse anti-desmoglein-2 antibody (sc-80663, Santa Cruz Biotechnology), rabbit anti-galectin-3 antibody (ab76245, Abcam), rabbit anti-galectin-3 binding protein (G3BP) antibody (ab217572, Abcam), rabbit anti-clusterin antibody (42143S, Cell Signaling Technology, MA, USA), and mouse anti-beta-actin (ACTB) antibody (sc-47778, Santa Cruz Biotechnology).

Techniques: Western Blot

Journal: Aging (Albany NY)

Article Title: Clusterin exerts a cytoprotective and antioxidant effect in human osteoarthritic cartilage

doi: 10.18632/aging.103310

Figure Lengend Snippet: Effects of CLU silencing on viability, proliferation/death and differentiation of cultured OA chondrocytes. ( A ) CLU mRNA and protein expression after 48 and 72 hours, respectively, in scramble (control) and siCLU transfected OA-HACs. ( B ) Cell viability by MTT assay after 24, 48 and 72 hours of scramble and siCLU transfected OA-HACs. ( C ) Representative immunocytochemical images and bar graphs showing Ki67 + and p21 + nuclei in cultured OA-HACs and ( D ) cleaved caspase-3 + cytospinned OA-HACs after 72 hours of silencing. ( E ) Gene array shows the modulation of NFkB and Stat-3-related genes in siCLU transfected OA-HACs compared with scramble after 48 hours of silencing. Transcript levels were normalized on GAPDH (internal reference gene). ( F ) mRNA levels by Real-Time PCR of COL2A1, ACAN, MMP13, COL10A1, and ( G ) representative blot and densitometric analysis of MMP13 and COL10A1 in scramble and CLU-silenced OA-HACs. Data are expressed as mean values ± SEM of three independent experiments (each using pooled samples from n=3 OA donors) performed in triplicate. Abbreviations: OA-HACs, OA-Human articular chondrocytes; OD, optical density unit; RLU, relative light unit. Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After separation by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were blotted to nitrocellulose transfer membranes and incubated with rabbit monoclonal anti-CLU (Cell Signaling Technology; 1:150, O/N), rabbit polyclonal anti-MMP13 antibody (Abcam; 1:500, O/N) and anti-NOX4 (Santa Cruz Biotechnology, USA; 1:200, O/N) and rabbit monoclonal anti-collagen X (Abcam; 1:1000, O/N).

Techniques: Cell Culture, Expressing, Transfection, MTT Assay, Real-time Polymerase Chain Reaction