Journal: Communications Biology

Article Title: Succinylation regulates boar sperm linear motility via reprogramming glucose metabolism

doi: 10.1038/s42003-025-08775-5

Figure Lengend Snippet: a Western blotting of VDAC3 in boar sperm. Protein loading was normalized using α-tubulin . b , c Following 3 h of incubation with HG or LG extender, VDAC3 protein bound to anti-PKM2 antibody was immunoprecipitated from boar sperm lysates. Immunoprecipitated VDAC3 was verified by Western blotting using a VDAC3 antibody ( b ). Quantitative expression of precipitated VDAC3 compared with the input generated from western blotting ( c ). Data are expressed as mean ± SD (n = 3). d , e Following 3 h incubation with HG extender, LG extender, HG + 3 mM SA, or LG + 100 μM RES, mitochondrial membrane permeability was assessed by measuring the opening of the MPTP. Flow cytometric analysis of sperm mitochondrial permeability transition pore opening after 3 h of incubation was performed ( d ). The fluorescence intensity of stained sperm was analyzed, with higher fluorescence intensity indicating lower MPTP opening ( e ). Data are expressed as mean ± SD (n = 6). f , g After 3 h of treatment with HG extender, LG extender, HG + 3 mM SA, or LG + 100 μM RES, changes in sperm mitochondrial membrane potential were evaluated. Flow cytometric analysis of sperm mitochondrial membrane potential after 3 h of incubation was conducted ( f ), and the percentage of sperm with high mitochondrial membrane potential was determined ( g ). Data are expressed as mean ± SD (n = 4). Effects of HG extender, LG extender, HG + 3 mM SA, or LG + 100 μM RES treatment on sperm mitochondrial complex IV enzyme activity ( h ) and ATP levels were assessed after 3 h of incubation ( i ). Data are expressed as mean ± SD (n = 6). a-c : Significant differences ( p < 0.05) between treatments. FS fresh semen, HG high glucose, LG low glucose, RES resveratrol, SA succinic acid.

Article Snippet: The membrane was incubated overnight at 4 °C with the primary antibody diluted in TBST containing 1% BSA, primary antibody including anti-succinyllysine (PTM-401, 1:1000, PTM BIO, Hangzhou, China), PKM2 (A20991, 1:1000 ABclonal, Wuhan, China), SIRT5 (bs-9456R, 1:1000, Bioss, Beijing, China), VDAC3 (bs-7647R, 1:2000, Bioss, Beijing, China), ODF2 (A3607, 1:500, ABclonal, Wuhan, China), NDUFA7 (A8441, 1:500, ABclonal, Wuhan, China) and α-Tubulin (ab7291, 1:10,000, Abcom, Shanghai, China).

Techniques: Western Blot, Incubation, Immunoprecipitation, Expressing, Generated, Membrane, Permeability, Fluorescence, Staining, Activity Assay

Journal: Communications Biology

Article Title: Succinylation regulates boar sperm linear motility via reprogramming glucose metabolism

doi: 10.1038/s42003-025-08775-5

Figure Lengend Snippet: Succinylation of PKM2 reduces its enzymatic activity, inhibits the glycolysis pathway in sperm, and enhances enzymes activity involved in the NADPH-producing pentose phosphate pathway. The succinylation-mediated mitochondrial translocation of PKM2 in boar sperm increases mitochondrial permeability through interaction with VDAC3, thereby stimulating OXPHOS to generate ATP and sustain high linear sperm motility. Furthermore, Sirt5 modulates PKM2 succinylation levels through its desuccinylase activity to maintain an optimal equilibrium between glycolysis pathway and OXPHOS. G6PD Glucose-6-phosphate dehydrogenase, 6PGD 6-Phosphogluconate dehydrogenase, 6-PG: 6-Phosphogluconate, R-5-P Ribose-5-phosphate, NADPH Nicotinamide adenine dinucleotide phosphate (reduced form), F-6-P Fructose-6-phosphate, F-1,6-P Fructose-1,6-bisphosphate, HK Hexokinase, PFK Phosphofructokinase, LDH Lactate dehydrogenase, ETC Electron transport chain, Sirt5 sirtuin 5, OXPHOS oxidative phosphorylation, VDAC3 voltage-dependent anion Channel protein 3.

Article Snippet: The membrane was incubated overnight at 4 °C with the primary antibody diluted in TBST containing 1% BSA, primary antibody including anti-succinyllysine (PTM-401, 1:1000, PTM BIO, Hangzhou, China), PKM2 (A20991, 1:1000 ABclonal, Wuhan, China), SIRT5 (bs-9456R, 1:1000, Bioss, Beijing, China), VDAC3 (bs-7647R, 1:2000, Bioss, Beijing, China), ODF2 (A3607, 1:500, ABclonal, Wuhan, China), NDUFA7 (A8441, 1:500, ABclonal, Wuhan, China) and α-Tubulin (ab7291, 1:10,000, Abcom, Shanghai, China).

Techniques: Activity Assay, Translocation Assay, Permeability, Phospho-proteomics

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: KEGG pathway clustering analysis of different comparison groups, the PPI network of differentially lactylated proteins in the pathways of interest, and verification of Vdac3 protein lactylation modifications. (A) Heatmap of lactylation sites for 12 key proteins. Each row represents a differentially modification site; each column represents a sample. Red indicates a high-level modification, blue indicates a low-level modification, and grey indicates sites that cannot be quantified in the corresponding samples. (B) The horizontal axis depicts different comparison groups, whereas the vertical axis indicates the enriched KEGG pathways. The colored blocks represent the functional descriptions of differentially expressed modified proteins enriched in each comparison group. Blue signifies high enrichment significance, whereas blue-white signifies low enrichment significance. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) PPI network of differentially lactylated proteins involved in the 4-VO group and sham group in the five pathways of interest. (D) PPI network in the 4-VO group and 4-VO + EA group. The color of the outer circle indicates the corresponding KEGG pathway of the protein, whereas the shape within the circle denotes the upregulation or downregulation of the modification sites contained within the protein. A diamond shape signifies proteins with upregulated modification sites, a downward arrow indicates proteins with downregulated modification sites, and a circle signifies proteins with upregulated and downregulated modification sites. The red box indicates the Vdac3 protein. (E) The IP experiment verified the lactylation modification level of Vdac3. (F) Quantification was performed using ImageJ software, and the values are expressed as the mean with the corresponding standard error from three replicate experiments ( n = 3, compared with the sham group, ## p < 0.01; compared with the 4-VO group, ** p < 0.01). 4-VO, four-vessel occlusion; EA, electroacupuncture; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction; IP, immunoprecipitation; Vdac3, mitochondrial voltage-dependent anion channel protein3.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Comparison, Modification, Functional Assay, Software, Immunoprecipitation

Journal: Frontiers in Neurology

Article Title: Effect of electroacupuncture on hippocampal protein lactylation in a rat model of vascular dementia

doi: 10.3389/fneur.2025.1629474

Figure Lengend Snippet: Analysis of lactylation site motifs in different groups and representative mass spectra of key proteins. (A) A heatmap of amino acids adjacent to lysine lactylation sites, with a green/red scale (−5 to 5) indicating the frequency of amino acid detection; red indicates high frequency; green indicates low frequency. The height ratio of amino acid abbreviation letters at specific positions reflects motif characteristics, with a % difference greater than 0 indicating a higher frequency of that amino acid at that position compared to the background, and less than 0 indicating a lower frequency. (B) Four conserved motif sites of lysine. (C) Sequence motif map of lactylation modification, 10 amino acids upstream and downstream of the modification site were selected, and the vertical axis indicates the difference between the proportion of the various amino acids at the same site in the experimental data set and the reference background, that is, percentage difference (%difference). The height ratio of amino acid abbreviation letters at specific positions represents the motif characteristics, and a %difference greater than 0 indicates that the frequency of the amino acid at this position is higher than the background. Values lower than 0 indicate that the frequency of the amino acid at this position is lower than the background. (D–F) The lactylation site of Vdac3 was identified by mass spectrometry. PPI, protein–protein interaction; 4-VO, four-vessel occlusion; EA, electroacupuncture.

Article Snippet: One milliliter of total protein extract was pre-cleared with 40 μL of protein A/G agarose beads by rotation at 4 °C for 1 h. Subsequently, the supernatant was incubated with 5 μL of Vdac3 antibody (A04802-2, BosterBio, China) overnight at 4 °C with rotation.

Techniques: Sequencing, Modification, Mass Spectrometry