Journal: bioRxiv

Article Title: Ketone body β-hydroxybutyrate restores neuronal Tau proteostasis via ketolysis-independent mechanism

doi: 10.64898/2026.01.30.702936

Figure Lengend Snippet: (A) Normalized protein abundances of Tau and Tubulin interactor OPTN from the interactome study. * p < 0.05 by linear regression model. (B) Representative Western blot of input lysate and co-IP with HT7 (hTau) in HEK293T cells overexpressing OPTN-eGFP and either mScarlet-Tau WT or mScarlet-Tau V337M , treated with βHB. IP, immunoprecipitation; IB, immunoblot; Tau5, total Tau. (C) Representative Western blot of input lysate and IP with HT7 (hTau) in HEK293T cells overexpressing mScarlet-Tau V337M , treated with βHB for 1 hr. IP, immunoprecipitation; IB, immunoblot; DAKO, total Tau; Ub, ubiquitin. (D) Schematic of Ub-dependent recognition of Tau at the autophagosome membrane by the LC3B-adapter protein OPTN. (E) Normalized protein abundances of Tau and Tubulin interactor LC3B-II. # p < 0.05, ## p < 0.01, #### p < 0.0001 by pairwise limma test. (F) Graphic illustrating mCherry-GFP-LC3B construct. mCherry was pseudocolored to magenta. APG, autophagosome; AL, autolysosome. (G-H) Representative immunofluorescent images (G) and quantification of autophagic vesicles (H) in primary neurons transfected with lenti-mCherry-GFP-LC3B and treated with βHB for 1 hr. Magenta-only vesicles were counted as autolysosomes, and double-positive bright green and magenta vesicles (white) were counted as autophagosomes. Each point represents an individual Map2+ neuron, with cells in the same well stacked into one column. Thick, color-coded bars represent the well mean (n = 8-15 cells/well), and black bars represent the overall group mean ± SD (n = 3 wells/group, from separate batches). Scale bar: 10 μm. (I) Quantification of percent of Tau secreted into the conditioned media (CM) in primary neurons infected with either lenti-shScramble or lenti-shOPTN for >5 days and treated with βHB for 1 hr. Value calculated by Tau levels in the CM divided by the sum of the CM Tau and intracellular (lysate) Tau, measured by ELISA. Each point represents one independent well, normalized to the control (shScramble NaCl) wells from its respective plate. (J-K) Representative immunofluorescent images (J) and quantification of Halo-Tau P301L (pink) co-localized with lysosomes (LysoTracker, green) in Map2+ neurons (K) co-transfected with lenti-Halo-Tau P301L and either lenti-shScramble or lenti-shOPTN for 5 days then labeled and co-treated with βHB and LysoTracker for 1 hr. Values represent the area of Halo-Tau co-localized to lysosomes over the total Halo-Tau area within a single neuron. Each point represents an individual Map2+ neuron, with cells in the same well stacked into one column. Thick, color-coded bars represent the well mean (n = 10-20 cells/well), and black bars represent the overall group mean ± SD (n = 3 wells/group, from separate batches). Scale bar: 10 μm. Data for A, E, and I are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant by linear mixed-effects model with Tukey post-hoc test (H, K) or by Šídák’s multiple comparisons test (I).

Article Snippet: Primary neurons were plated at a density of 600K per mL onto PDL-coated 12-well plates or coverslips and were infected on DIV3 with the homemade lentivirus for 5 days. shOPTN efficacy was validated via Western blot with anti-OPTN antibody (Proteintech, #10837-1-AP).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Construct, Transfection, Infection, Enzyme-linked Immunosorbent Assay, Control, Labeling

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

Journal: Alzheimer's Research & Therapy

Article Title: Genetic and clinical landscape of Chinese frontotemporal dementia: dominance of TBK1 and OPTN mutations

doi: 10.1186/s13195-024-01493-w

Figure Lengend Snippet: Biochemical characterization of TBK1 variants. A Domain structure and OPTN-binding site of TBK1 protein. The variants identified in this study are indicated. B Top five lanes: HEK293T cells expressing Flag-TBK1 wild-type and indicated mutants were co-transfected with mCherry-OPTN for 48 h before lysis. The binding of TBK1 variants was analyzed via immunoblot (IB). The asterisk points to OPTN pS177 bands. Bottom two lanes: Coimmunoprecipitation (co-IP) of Flag-TBK1 (wild-type and indicated mutants) and mCherry-OPTN wild-type from HEK293T cell lysates. C Phosphorylation of OPTN S177 was confirmed by IB using the pS177 OPTN antibody generated by immunoGlobe GmbH. EV: empty vector. D Cell lysates were subjected to immunoblot analysis and active TBK1 (pS172) was detected with a phosphospecific antibody (pS172; no. 5483; Cell Signaling Technology). E Co-immunoprecipitation (Co-IP) of Flag-TBK1 wild-type and the indicated mutants with mCherry-OPTN wild-type from HEK293T cells using an antibody to Flag. Co-immunoprecipitated proteins were analyzed by immunoblotting (IB) with the Flag and mCherry antibodies

Article Snippet: C Phosphorylation of OPTN S177 was confirmed by IB using the pS177 OPTN antibody generated by immunoGlobe GmbH.

Techniques: Binding Assay, Expressing, Transfection, Lysis, Western Blot, Co-Immunoprecipitation Assay, Phospho-proteomics, Generated, Plasmid Preparation, Immunoprecipitation