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