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: Toxics

Article Title: Effects of Short-Term Exposure to Polystyrene Nanoplastics on the Nervous System: Calcium Homeostasis, BDNF and Synaptic Plasticity

doi: 10.3390/toxics14020178

Figure Lengend Snippet: Role of BDNF in the mechanism of synaptic plasticity impairment. All indicators were detected in neurons from the control and PS-NPs intervention groups. ( A ) Western blot analysis of CREB, BDNF, Cav3.1, and CaMKIIα in neurons, with GAPDH as the loading control. ( B ) Protein expression levels of CREB in the neurons. n = 3. ( C ) Protein expression levels of BDNF in the neurons. n = 3. ( D ) Calcium ion content in the neurons. n = 6. ( E ) Protein expression levels of the calcium channel Cav3.1 in neurons. ( n = 3). ( F ) Protein expression levels of CaMKIIα in neurons. n = 3. (○) represents the individual samples within each group. Quantitative results were normalized, and the data in the bar charts are presented as mean ± SD. ** p < 0.01 or *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skim milk (Beyotime, Shanghai, China, P0216-1500 g) and incubated overnight at 4 °C with specific primary antibodies, including Cav3.1 (1:2000, NBP2-59322, Novus, Lone Tree, CO, USA), CaMKIIα (1:2000, 3357S, CST, Danvers, MA, USA), CREB (1:2000, 12208-1-AP, Proteintech, Rosemont, IL, USA), BDNF (1:1000, ab226843, abcam, Cambridge, UK), PSD95 (1:1000, 2507S, CST), SYN (1:2000, 17785-1-AP, Proteintech), ProBDNF (1:1000, 28205-1-AP, Proteintech), and GAPDH (1:2000, UM4002, YouKang, Tianjin, China).

Techniques: Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: EOLA1, a novel mitochondria-localized protein critical for heart functions via regulating mitochondrial translation

doi: 10.64898/2026.01.12.699056

Figure Lengend Snippet: a, Mitochondrial proteins predicted by TargetP 2.0 in human and mouse (left), and their overlap with MitoCarta 3.0 annotations (right). b, Fluorescence analysis of the subcellular location of human EOLA1 protein (red), and HSP60 (green) was used as a marker for mitochondria. The exogenously expressed EOLA1 was tagged with a C-terminal FLAG and HA tandem epitope. c, Western blotting analysis of EOLA1 in the subcellular fractions of PLC/PRF/5 ( Homo sapiens , left) and B16-F10 melanoma cells ( Mus musculus , right). Tubulin was used as a cytoplasmic marker, Histone H1.2 as a nuclear marker, and HSP60 as a mitochondrial marker. d, Determination of EOLA1 sub-mitochondrial localization by Proteinase K digestion assay using purified mitochondria from HEK293T cells. PK, Protease K; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. e, f, Seahorse analysis of WT and Eola1 KO in B16-F10 melanoma cells ( e ) and HL-1 cells ( f ). Quantification of the basal respiration, maximal respiration, and spare respiratory capacity was performed for the indicated cells. g-i, Electron microscopy images ( g ) of WT cells (left) and Eola1 knockout cells (right). Higher-magnification views are presented in panel h . The relative circularity ratio of mitochondria was quantified ( i ). Mito: Mitochondria, N: nucleus. Note: e , f , One-way ANOVA with Tukey’s test. i , two-tailed unpaired Student’s t-tests. ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. e , f , Data were presented as mean ± SD (n=6).

Article Snippet: The corresponding antibodies included anti-EOLA1 antibody (1:5000, CUSABIO, CSB-PA837446LA01HU), anti-HSP60 antibody (1:3000, Proteintech, 15282-1-AP), anti-Tom20 antibody (1:1000, ABclonal, A19403), anti-α-Tubulin antibody (1:3000, Proteintech, 80762-1-RR), anti-HistoneH1.2 antibody (1:1000, Proteintech, 19649-1-AP), anti-TUFM antibody (1:1000, SAB, 34669), anti-FLAG antibody (1:1000, Sigma, F1804), anti-MRPS15 antibody (1:1000, Proteintech, 17006-1-AP), anti-MRPL11 antibody (1:1000, Proteintech, 15543-1-AP), anti-ATP6 antibody (1:500, SAB, 31464), Anti-COX1 antibody (1:500, ABclonal, A23123), anti-MT-ND5 antibody (1:500, Proteintech, 55410-1-AP), anti-β-actin antibody (SAB, 52901), and anti-GAPDH antibody (1:5000, Proteintech, 10494-1-AP).

Techniques: Fluorescence, Marker, Western Blot, Purification, Membrane, Electron Microscopy, Knock-Out, Two Tailed Test

Journal: Nutrients

Article Title: Integrated Proteomics and Metabolomics Reveal the Direct Hepatic Protection of Propionate Against Alcoholic Liver Disease via the RGN-PPARα Pathway

doi: 10.3390/nu18050872

Figure Lengend Snippet: The effect of PA on lipid accumulation in ALD mice. ( A ) Schematic of the acute alcoholic liver injury model in mice with propionate treatment. ( B , C ) Serum ALT and AST levels in mice. ( D , E ) H&E and Oil Red O staining of liver tissues (scale bar: 50 μm). ( F ) The relative area of Oil Red O staining. ( G , H ) Liver levels of TC and TG in mice. ( I – L ) Serum concentrations of TC, TG, HDL-C and LDL-C in mice. ( M ) Representative Western blot analysis and ( N ) quantification of ACC1, SREBP1 and CPT1A protein expression in liver tissues normalized to β-actin (n = 4). The data are presented as means ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies, including β-actin (#66009-1-Ig), CPT1A (#15184-1-AP), SREBP-1 (#14088-1-AP), ACC1 (#21923-1-AP), ACOX1 (#83731-2-RR), PPARα (#66826-1-Ig) and RGN (#17947-1-AP), were obtained from Proteintech Group (Wuhan, China).

Techniques: Staining, Western Blot, Expressing

Journal: Nutrients

Article Title: Integrated Proteomics and Metabolomics Reveal the Direct Hepatic Protection of Propionate Against Alcoholic Liver Disease via the RGN-PPARα Pathway

doi: 10.3390/nu18050872

Figure Lengend Snippet: Propionate suppressed cell injury and lipid accumulation in vitro. ( A ) Changes in AML-12 cell viability after a series of doses of ethanol challenge. ( B ) Changes in AML-12 cell viability after a series of concentrations of OA incubation. ( C ) Effects of different concentrations of propionate on the viability of AML-12 cells. ( D ) Propionate restored AML-12 cells’ viability following co-treatment with 200 mM ethanol and 0.5 mmol/L OA. ( E ) Representative views of Oil Red O staining of AML-12 cells (scale bars: 100 μm and 50 μm). ( F ) Representative images of Western blot analysis and ( G ) quantification of ACC1, SREBP1 and CPT1A protein expression in AML-12 cells normalized to β-actin (n = 3). The data are presented as means ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies, including β-actin (#66009-1-Ig), CPT1A (#15184-1-AP), SREBP-1 (#14088-1-AP), ACC1 (#21923-1-AP), ACOX1 (#83731-2-RR), PPARα (#66826-1-Ig) and RGN (#17947-1-AP), were obtained from Proteintech Group (Wuhan, China).

Techniques: In Vitro, Incubation, Staining, Western Blot, Expressing