Journal: Cells

Article Title: Age-Dependent Dysregulation of APP in Neuronal and Skin Cells from Fragile X Individuals

doi: 10.3390/cells12050758

Figure Lengend Snippet: APP processing in human TDI and FXS fibroblasts. Representative Western blot showing sAPPα, ADAM10, and FMRP in TDI and FXS individuals stratified according to the age: ( A ) <20 years (TDI n = 10; FXS n = 10), ( B ) between 20 and 30 years (TDI n = 5; FXS n = 11) and ( C ) >30 years old (TDI n = 4; FXS n = 11). The bar plots show the quantification of sAPPα and ADAM10 levels in TDI and FXS fibroblast cell lines normalized to Coomassie staining and Vinculin, respectively. Error bars represent the SEM (* p < 0.05, Mann–Whitney test). ADAM10 and FMRP proteins were detected on separate blots, each with its own control Vinculin.

Article Snippet: Membranes were incubated using the following specific antibodies, including mouse anti-puromycin (1:500, DSHB), mouse anti-Vinculin (1:2000, Merck), mouse anti-GAPDH (1:2000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-APP (1:2000, Merck), rabbit anti-ADAM10 (1:500, Abcam, Cambridge, UK), mouse anti-sAPPα (1:500, IBL America, Minneapolis, MN, USA), rabbit anti-OCT3/4 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-MAP2 (1:2000, Merck), mouse anti-Nestin (1:1000 Santa Cruz Biotechnology), mouse anti-SAP97 (1:1000, ENZO Life Sciences, Farmingdale, NY, USA) and rabbit anti-FMRP (1:1000, produced in house PZ1 [ ]), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:5000, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Protective variant in PLCγ2 mitigates Alzheimer’s disease-associated pathologies via enhancing beneficial microglia functions

doi: 10.1101/2024.09.12.612207

Figure Lengend Snippet: A) X-34-positive β-amyloid plaque count (**p=0.005) and coverage (**p=0.006, total plaque area, % of whole analyzed area) are decreased in the entorhinal cortex of the APP/PS1xPLCγ2-P522R (A+/P ki/ki ) mice as compared to the APP/PS1 (A+/P wt/wt ) mice. Size (µm 2 ) of the individual plaques does not differ between the genotypes. n(A+/P wt/wt )=4, n(A+/P ki/ki )=6. B) Insoluble, but not soluble Aβ40 and –42 levels are slightly, but not significantly lower in the temporo-occipital cortex of the A+/P ki/ki mice as compared to the A+/P wt/wt mice. Aβ40 and –42 levels are normalized to the total protein concentration in the same sample. n(A+/P wt/wt )=4, n(A+/P ki/ki )=4. C) Representative Western blots and corresponding quantification show no differences in the levels of full-length APP (APPtot, normalized to GAPDH), APP C-terminal fragments (C99 and C83, normalized to APPtot), or soluble APPα and –β (sAPPα, sAPPβ, normalized to APPtot) species in the temporo-occipital cortex of the A+/P ki/ki and the A+/P wt/wt mice. n(A+/P wt/wt )=4, n(A+/P ki/ki )=4. Scale bars in the representative immunofluorescent images are 657µm for the whole area and 164µm for the zoomed view. Unpaired samples T-test. All data are presented as mean ± SEM. Each datapoint represents an individual mouse.

Article Snippet: Proteins were detected from the blots using the following primary antibodies diluted in 1x TBST and incubated overnight at + 4 °C: mouse anti-β-amyloid 1:1000 (6E10, #SIG-39320, BioLegend), rabbit anti-sAPPβ 1:100 (#18957, IBL America), mouse anti-APP N-terminal 1:1000 (22C11, #MAB348, Merck), rabbit anti-APP C-terminal 1:1000 (#A8717, Merck), and mouse anti-GAPDH 1:10000 (#ab8226, Abcam).

Techniques: Protein Concentration, Western Blot