Journal: Nature Biomedical Engineering
Article Title: Amelioration of Alzheimer’s disease pathology by mitophagy inducers identified via machine learning and a cross-species workflow
doi: 10.1038/s41551-021-00819-5
Figure Lengend Snippet: a , Effects of Kaem and Rhap on protein levels of full-length APP (FL-APP), CTF-α and CTF-β in hippocampal tissues from 3xTg AD mice (n = 3 per group). b , c , Quantification of phosphorylated Tau sites (Thr231) and total Tau/GAPDH in hippocampal tissues from 3xTg AD mice (n = 3 biologically independent samples). d , e , Effects of Kaem and Rhap on microglial phagocytosis of Aβ plaques in hippocampal tissues from 3xTg AD mice. Data were quantified from 3 random images/mouse from a total of 3 mice ( d ). Aβ plaques are shown in green (6E10 antibody) and microglia (anti-Iba1 antibody) are in red ( e ). f , Western blot results showing the effects of Kaem and Rhap on the levels of proteins involved in mitophagy (PINK1, Parkin, OPTN, p-ULK1-Ser555 and ULK1) and substrates of autophagy (p62 and LC3-II/I) in the hippocampal tissues of the mice (n = 3 mice per group). g – l , Quantification of Western blot data in ( f ), n = 3 biologically independent samples. se: short-exposure; le: long-exposure. m , Western blot results showing the effects of Kaem and Rhap on the levels of proteins involved in OXPHOS in the hippocampal tissues of the mice (n = 3 mice per group). n , Effects of Kaem and Rhap on autophagy induction using a HeLa cell line stably expressing GFP-LC3 following Kaem (10 μM) or Rhap (10 μM) treatment for 12 h before imaging. o , p , Western blot data (o) with semi-quantification (p) showing the effects of Kaem and Rhap on levels of LC3-II/I in the HEK293 cells (n = 3 biologically independent samples). All quantitative data shown in mean ± S.E.M. One-way ANOVA followed by Šidák’s multiple comparisons test ( b – d , g – l , p ) was used for data analysis. NS, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Original unprocessed western blots for a , f , m , o are available in Source Data Figs. 2–4.
Article Snippet: Antibodies used were as follows (all from Cell Signaling Technology, unless otherwise stated): PINK1 antibody (catalog no. ab75487, Abcam; no. A7131, ABclonal), Parkin antibody (no. NB100–91921, Novus), FUNDC1 antibody (no. ab74834, Abcam), LC3B antibody (no. NB100–2220, Novus), Beclin1 antibody (no. 3495s), phospho-DRP1 antibody (no. S616), DRP1 antibody (no. 8570s), p62 antibody (no. 8025s), MFN2 antibody (no. 94823s), phospho-ULK1 antibody (no. 5869s), ULK1 antibody (no. 6439s), AMBRA1 antibody (no. 24907s), OPTN antibody (no. A1845, ABclonal), Tau antibody (no. 46687s), p-Tau-thr181 (no. 12285s), p-Tau-thr231 (no. ab151559, Abcam), p-Tau-ser202/thr205 (no. MN1020, ThermoFisher), p-Tau-thr217 (no. 44–744, ThermoFisher), beta amyloid polyclonal antibody (no. 51–2700, ThermoFisher), Parkin Rabbit pAb (no. A0968, ABclonal), Tim23 antibody (no. 611222, BD Biosciences), Total OXPHOS Rodent WB Antibody Cocktail (no. ab110413, Abcam) and β-actin antibody (no. A5441, Sigma).
Techniques: Western Blot, Stable Transfection, Expressing, Imaging
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