Fig. 5

The protein Arl8b is upregulated in hippocampal tissues of 5xFAD mice. a Time-depended change of Arl8b protein abundance in hippocampal and cortical tissues of 5xFAD transgenic animals. LFC, log2 fold-change. b Hippocampal brain homogenates prepared from four 8-month-old 5xFAD (1 to 4) and control (ctrl; 1 to 4) mice were analyzed by SDS-PAGE and immunoblotting using anti-Arl8b. As a control, a tubulin (anti-alpha Tubulin, #T6074) immunoblot was performed. c Quantification of Arl8b expression in relation to tubulin using band intensities of immunoblots in b. Relative intensity values (mean ± SD) are shown for AD (n = 4) and Ctrl (n = 4) mice. Statistical significance was assessed between AD and Ctrl mice using an unpaired, two-tailed t-test (*, p = 0.0137). d Immunofluorescence analysis of 5xFAD mouse (8 months) brain slices using AlexaFluor594-labelled 6E10 antibody (red); an anti-Arl8b antibody combined with an AlexaFluor647-labelled anti-rabbit IgG (turquoise) was applied to detect Arl8b. The scale bar shown in the 6E10 image also applies to the Arl8b and merge image. The picture on the right shows a magnification (magnif.) of an area indicated in the merged picture. e Brain slices of 8-month-old 5xFAD mice were stained with the primary antibodies indicated in the images. For detection with 352 and Lamp1 antibodies, an AlexaFluor594-labelled anti-mouse IgG (red) was used; for Arl8b detection, an AlexaFluor647-labelled anti-rabbit IgG (turquoise) was applied. Antibody 352 specifically recognizes Aβ42 fibrillar aggregates [30]. f Pearson correlation between the volumes of Arl8b accumulations and 6E10-stained amyloid-beta plaques in hippocampus of 2- (H2), 5- (H5), and 8- (H8) month-old 5xFAD mice. A total of 60 plaques were analyzed in brain slices derived from 5- and 8-month-old 5xFAD mice. For 2-month-old 5xFAD mice, less than 60 plaques were analyzed, since amyloid burden at this age is low. The statistical significance of the association between the volumes of Arl8b accumulations and 6E10-stained amyloid-β plaques was measured with a two-tailed t-test (*, p = 0.024; ****, p < 0.0001)