anti mouse apoe  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Human breast milk extracellular vesicles from donors with asthma differentially the modulate release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

doi: 10.64898/2026.03.02.709065

Figure Lengend Snippet: (A) Schematic representation of BM-EVs isolation and characterization. BM was collected from 3-4 months post-partum mothers with and without asthma diagnosis. BM-EVs were isolated by SEC (qEV original columns, Izon Science Ltd) and analyzed for size, concentration and zeta potential through TRPS using the qNano Gold instrument (Izon Science Ltd.). BM-EVs were concentrated for protein yield quantification through MicroBCA Protein Assay kit (Thermo Scientific), Western blot analysis for exosome and microvesicles markers and hASM cells treatment for cytokines release analysis. (B) TEM showing exosomal morphology and approximate size of BM-EVs (scale bar 100[nm). (C) Western blotting was performed (12% SDS-PAGE) and Ponceau S staining used as a loading control. Proteins enriched in exosomes (CD9, CD81, CD63, TSG101, HSP70 and Flotillin-1), in microvesicles (MMP2 and ARF6), and lipoproteins (APO-A1) were analysed. F7 to 9 were considered exosome-rich while microvesicles and lipoprotein-poor. CL: cell lysate from breast tissue, M: marker lane.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-TSG101 (T5701, Sigma-Aldrich Co, 1:200), rabbit polyclonal anti-CD63 (SAB4301607, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-CD81 (sc-166029, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-CD9 (CBL162, Sigma-Aldrich Co, 1:100), rabbit polyclonal anti-Flotillin-1 (F1180, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-heat shock protein 70 (HSP70) (H5147, Sigma-Aldrich Co, 1:500), mouse monoclonal anti-ARF6 (sc-7971, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-MMP2 (sc-13595, Santa Cruz Biotechnology, 1:200) and mouse monoclonal anti-Apolipoprotein A1 (Apo-A1) (0650-0050, Bio-Rad Laboratories, 1:200).

Techniques: Isolation, Biomarker Discovery, Concentration Assay, Zeta Potential Analyzer, Western Blot, SDS Page, Staining, Control, Marker

Journal: bioRxiv

Article Title: Human breast milk extracellular vesicles from donors with asthma differentially the modulate release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

doi: 10.64898/2026.03.02.709065

Figure Lengend Snippet: (A) Equal amounts of protein (5 µg/mL) of BM-EVs from mothers with and without asthma were subjected to SDS-PAGE (12% SDS) and the expression of proteins enriched in exosomes [CD81 (22 kDa), CD63 (28 kDa), CD9 (25 kDa), Flotillin-1 (48 kDa), TSG101 (46 kDa), HSP70 (70 kDa)], in microvesicles [MMP2 (63kDa)], and in lipoproteins [APO-A1 (25kDa)] were quantified. Ponceau or Coomassie staining was used as a loading control to normalize protein content for all markers. (B) CD81 levels remained unchanged between the groups, (C) while asthma BM-EVs expressed ∼86% lower CD63 (p=0.0224) and (D) ∼ 24% lower CD9 (p=0.0646). (E) Flotillin-1 expression was lower by ∼40% in asthma BM-EVs compared to control (p=0.0196). (F) TSG101 levels remained unchanged, (G) while HSP70 was ∼69% lower in the asthma BM-EV (p=0.08). (H) APO-A1 was used as a purity control to investigate the presence of lipoproteins in the samples and did not show difference between groups. BM-EVs did not show any expression for MMP2 protein. Data were analyzed using an unpaired Student’s t-test with p<0.05 considered as significant and expressed as mean ± standard error (N=5).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-TSG101 (T5701, Sigma-Aldrich Co, 1:200), rabbit polyclonal anti-CD63 (SAB4301607, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-CD81 (sc-166029, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-CD9 (CBL162, Sigma-Aldrich Co, 1:100), rabbit polyclonal anti-Flotillin-1 (F1180, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-heat shock protein 70 (HSP70) (H5147, Sigma-Aldrich Co, 1:500), mouse monoclonal anti-ARF6 (sc-7971, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-MMP2 (sc-13595, Santa Cruz Biotechnology, 1:200) and mouse monoclonal anti-Apolipoprotein A1 (Apo-A1) (0650-0050, Bio-Rad Laboratories, 1:200).

Techniques: SDS Page, Expressing, Staining, Control

Journal: Journal of Lipid Research

Article Title: Amino acid residues L261, W264, F265, L268, and V269 of apolipoprotein E4 critically regulate adipose tissue metabolism

doi: 10.1016/j.jlr.2025.100958

Figure Lengend Snippet: Effects of APOE4 and APOE4mut1 on 3T3-L1 adipocyte differentiation. A: Representative Western blot analysis for APOE4, APOE4mut1 in samples of culture medium collected on the 10th day of 3T3-L1 cell differentiation as well as β-tubulin in the respective cell lysates. Samples for APOE were analysed on two separate blots. B: Semiquantitative analysis of APOE4 and APOE4mut1 levels shown in the blots of panel A, normalized for β-tubulin levels (C–E) Representative images at 10× magnification of differentiated 3T3-L1 adipocytes after Oil Red O staining. The cells were induced to differentiate after infection with: (C) AdGFP, (D) AdAPOE4, (E) AdAPOE4mut1. F: Relative absorbance of Oil Red O dye, eluted from cultures infected with recombinant adenovirus. The values were derived from the average of at least three cultures for each adenovirus used. The data were analyzed using one-way ANOVA, and the values are expressed as Mean ± SEM. G: Effect of APOE4 and APOE4mut1 on triglyceride accumulation in mature 3T3-L1 adipocytes. The values for each culture were normalized to the total protein levels of the cells present in the culture. The analysis of the results was performed using one-way ANOVA, and the values are presented as Mean ± SEM. H: mRNA expression levels of the lipogenic genes ppar γ , dgat1 , and fasn as well as ppar γ effector genes adiponectin , fabp4 , fabp5 , and lpl in differentiated 3T3-L1 adipocytes expressing APOE4 and APOE4mut1. The results were normalized to the expression levels of the housekeeping rps18 gene. The analysis was performed using two-way ANOVA, and the values are presented as Mean ± SEM. ∗∗ = P < 0.005 and ∗∗∗ = P < 0.0005, n = 3 per group. Unless indicated by a horizontal line between two groups, statistical significance refers to differences among all test groups.

Article Snippet: For the semiquantitative measurement of murine and human protein levels in the plasma and the lipoprotein fractions of the mice, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by Western blot, using the following antibodies: Rabbit anti-Human and anti-Mouse APOE (D7I9N, cat# 10197, Cell Signaling), Goat anti APOB-48/100 HRP (cat# K34005G, Meridian Life Science), Rabbit anti-Mouse β-tubulin(cat# # 2146, Cell Signaling), Rabbit anti-Mouse Cytc (cat# 4272, Cell Signaling), Rabbit anti-Mouse Ucp1 (E9Z2V, cat#72298, Cell Signaling), Rabbit anti-Mouse Cox4 (cat# 4844, Cell Signaling), and Goat anti-rabbit IgG-HRP (cat#7074, Cell Signaling).

Techniques: Western Blot, Cell Differentiation, Staining, Infection, Recombinant, Derivative Assay, Expressing

Journal: Journal of Lipid Research

Article Title: Amino acid residues L261, W264, F265, L268, and V269 of apolipoprotein E4 critically regulate adipose tissue metabolism

doi: 10.1016/j.jlr.2025.100958

Figure Lengend Snippet: Plasma lipid and glucose levels of C57BL/6 mice fed high-fat diet for 8 weeks and then infected with the adenoviruses AdGFP, AdAPOE4, and AdAPOE4mut1. A: Representative Western blot of human and murine APOE five days after infection of mice with the adenoviruses, (B) plasma total cholesterol, (C) plasma total triglyceride, and (D) plasma glucose levels five days after infection of mice with the adenoviruses. The results were analyzed using one-way ANOVA. Values are expressed as Mean ± SEM. ∗ = P < 0.05, ∗∗ = P < 0.005 and ∗∗∗ = P < 0.0005, n = 3 per group.

Article Snippet: For the semiquantitative measurement of murine and human protein levels in the plasma and the lipoprotein fractions of the mice, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by Western blot, using the following antibodies: Rabbit anti-Human and anti-Mouse APOE (D7I9N, cat# 10197, Cell Signaling), Goat anti APOB-48/100 HRP (cat# K34005G, Meridian Life Science), Rabbit anti-Mouse β-tubulin(cat# # 2146, Cell Signaling), Rabbit anti-Mouse Cytc (cat# 4272, Cell Signaling), Rabbit anti-Mouse Ucp1 (E9Z2V, cat#72298, Cell Signaling), Rabbit anti-Mouse Cox4 (cat# 4844, Cell Signaling), and Goat anti-rabbit IgG-HRP (cat#7074, Cell Signaling).

Techniques: Clinical Proteomics, Infection, Western Blot

Journal: Journal of Lipid Research

Article Title: Amino acid residues L261, W264, F265, L268, and V269 of apolipoprotein E4 critically regulate adipose tissue metabolism

doi: 10.1016/j.jlr.2025.100958

Figure Lengend Snippet: Plasma lipoprotein lipid levels of C57BL/6 mice fed high-fat diet for 8 weeks and then infected with the adenoviruses AdGFP, AdAPOE4, and AdAPOE4mut1. Lipoproteins were fractionated by KBr density gradient ultracentrifugation. A, C–E: Total cholesterol and (F–I) triglyceride levels of lipoprotein fractions 5 days after infection of mice with the adenoviruses. Panel B shows the murine APOE, human APOE4 and APOE4mut1 distribution among lipoprotein fractions. The results were analyzed using one-way ANOVA, and values are expressed as Mean ± SEM. ∗∗∗ = P < 0.0005, n = 3 per group. Statistical significance refers to differences among all test groups.

Article Snippet: For the semiquantitative measurement of murine and human protein levels in the plasma and the lipoprotein fractions of the mice, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by Western blot, using the following antibodies: Rabbit anti-Human and anti-Mouse APOE (D7I9N, cat# 10197, Cell Signaling), Goat anti APOB-48/100 HRP (cat# K34005G, Meridian Life Science), Rabbit anti-Mouse β-tubulin(cat# # 2146, Cell Signaling), Rabbit anti-Mouse Cytc (cat# 4272, Cell Signaling), Rabbit anti-Mouse Ucp1 (E9Z2V, cat#72298, Cell Signaling), Rabbit anti-Mouse Cox4 (cat# 4844, Cell Signaling), and Goat anti-rabbit IgG-HRP (cat#7074, Cell Signaling).

Techniques: Clinical Proteomics, Infection

Journal: Stroke and Vascular Neurology

Article Title: Enhanced liver X receptor signalling reduces brain injury and promotes tissue regeneration following experimental intracerebral haemorrhage: roles of microglia/macrophages

doi: 10.1136/svn-2023-002331

Figure Lengend Snippet: GW3965 upregulates LXR downstream-gene expression. (A) Representative images of confocal tile scan showing whole day 7 lesion labelled with DAPI (blue), GFAP (Cyan), ABCA1 (green) and Iba1 (red). Scale bar, 200 µm. (B) Representative image of a three-dimensional (3D) reconstruction of a perihaematomal region in the treatment group using Imaris. Scale bar, 3 µm. (C) Representative confocal images of perihaematomal region labelled with DAPI (blue), GFAP (cyan), ABCA1 (green) and Iba1 (red). The left corner within dotted lines depicts lesion centre. Scale bar, 100 µm. (D–F), Bar graphs comparing the ABCA1 + percentage (D), Iba1 + ABCA1 + over Iba1 + proportions (E) and GFAP + ABCA1 + over GFAP + proportions (F) between GW3965 treatment and DMSO control within the lesion region at day 7; the sample size was n=10 per group. (G) Representative images of confocal tile scan showing whole day 7 lesion labelled with DAPI (blue), Iba1 (cyan), GFAP (green) and ApoE (red). Scale bar, 200 µm. Scale bars are not equal. (H), Representative image of a 3D reconstruction of a perihaematomal region in the treatment group using Imaris. Scale bar, 7 µm. (I), Representative confocal images of perihaematomal region labelled with DAPI (blue), Iba1 (cyan), GFAP (green) and ApoE (red). The left corner within dotted lines is the lesion centre. Scale bar, 100 µm. (J–L), Bar graphs comparing the ApoE + percentage (J), GFAP + ApoE + over GFAP + proportions (K) and Iba1 + ApoE + over Iba1 + proportions (L) between GW3965 treatment and DMSO control within the lesion region at day 7; the sample size was n=10 per group. Significance is indicated as *p<0.05, **p<0.01, ****p<0.0001; two-tailed, unpaired t-test (D, F, K); or two-tailed, unpaired t-test with Welch’s correction (E); or Mann-Whitney U test (J, L). Bar graphs show individual data points and represent mean±SD. ApoE, apolipoprotein E; LXR, liver X receptor.

Article Snippet: After transferring to Hybond polyvinylidene difluoride (Amersham) membrane using a wet transfer system (Bio-Rad), membranes were blocked with 5% BSA and then probed overnight with the following primary antibodies: rabbit anti-human/mouse ABCA1 (1:1000; Cell Signaling, 96292), rabbit anti-mouse ApoE (1:1000; Cell Signaling, 49285), mouse anti-human/mouse IL-1β (1:1000; Cell Signaling, 12242), rabbit anti-mouse iNOS (1:1000; Cell Signaling, 13120), rabbit anti-human/mouse CD206 (1:1000; Cell Signaling, 24595), rabbit anti-human/mouse Arginase 1 (1:1000; Cell Signaling, 93668) and rabbit anti-mouse β-Actin (1:1000; Cell Signaling, 24595).

Techniques: Gene Expression, Control, Two Tailed Test, MANN-WHITNEY