Journal: Journal of Biological Chemistry

Article Title: Developmental Control of Apoptosis by the Immunophilin Aryl Hydrocarbon Receptor-interacting Protein (AIP) Involves Mitochondrial Import of the Survivin Protein

doi: 10.1074/jbc.m110.210120

Figure Lengend Snippet: FIGURE 6. Tom20 regulation of mitochondrial import of survivin. A, ali- quots of GST, GST-Tom20, or GST-Tom70 recombinant proteins were mixed with Raji cell extracts, and bead-bound material was analyzed by Western blotting. Bottom panel, Coomassie Blue staining of recombinant GST fusion proteins. B, Raji mitochondrial extract was immunoprecipitated with an anti- body to survivin or IgG, and proteins in pellets or supernatants (unbound) were analyzed by Western blotting. C, recombinant wild-type survivin or the survivin 1–141 mutant and GST-Tom20 beads were mixed with increasing concentration of recombinant AIP, and bead-bound proteins were analyzed by Western blotting (top panel). Bottom panel, GST or GST-Tom20 was incu- bated with recombinant AIP in the presence of increasing concentrations of survivin (up to 0.4 M), and bound proteins were analyzed by Western blot- ting. D, independently established clones (#) of HeLa cells stably transfected with control (Ctrl) or Tom70- or Tom20-directed shRNA were analyzed by Western blotting. E, HeLa cells were transfected with control, non-targeting (Ctrl) or Tom20-directed siRNA and analyzed by Western blotting. TCE, total cell extracts. F, clones of HeLa cells with stable knockdown of Tom70 or Tom20 were treated with 0.5 M staurosporine for 24 h and analyzed for cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. DataaremeanS.E.ofreplicates(n3).***,p0.0001.Twodifferentclones per conditions were tested with identical results in two independent experiments.

Article Snippet: Antibodies against AIP (Novus Biologicals), survivin (Novus Biologicals), Tom20 (Santa Cruz Biotechnology), Tom70 (Novus Biologicals), COX-IV (Clontech), Smac (Pro-Sci), -actin (Sigma), and Hsp90 (BD Biosciences) were used.

Techniques: Recombinant, Western Blot, Staining, Immunoprecipitation, Mutagenesis, Concentration Assay, Clone Assay, Stable Transfection, Transfection, Control, shRNA, Knockdown

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

Journal: Molecular nutrition & food research

Article Title: Lack of β, β-carotene -9’, 10’-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice

doi: 10.1002/mnfr.201600576

Figure Lengend Snippet: Functional categorization of proteins differentially expressed in hepatic mitochondria of BCO2 knockout (KO) vs. 129S6 wild type (WT) mice identified by spectrum counting. Values are means, n=3 mice/group with 4 technical replicates/mouse.

Article Snippet: Antibodies against fatty acid synthase (FASN) (catalog # 10624-2-Ap), ATP synthase α subunit 1 (ATP5A1) (catalog # 14676-1-AP), citrate synthase (catalog # 16131-1-AP), hydroxyacyl-coenzyme A dehydrogenase (HADH) (catalog # 19828-1-AP), succinate dehydrogenase α (SDHA) (catalog # , 14865-1-AP), and nicotinamide nucleotide transhydrogenase (NNT) (catalog # 13442-2-AP) were purchased from ProteinTech Group (Chicago, IL, USA); Antibodies against lysosome-associated membrane protein 1 (LAMP1) (catalog # sc-20011), catalase (catalog # sc-50508), NNT (catalog # sc-163154), glutathione reductase (GSR) (catalog # sc-133245) were purchased from Santa Cruz Biotech (Dallas, TX, USA); peroxisome proliferator-activated receptor α (PPARα) antibody (catalog # 101710) was purchased from Cayman (Ann Arbor, MI, USA); Antibodies against β-actin (catalog # 4967), phosphor-Thr172-AMP-activated protein kinase α (pT172-AMPKα) (catalog # 2535), and AMPKα (catalog # 2603) were purchased from Cell Signaling Technology (Danvers, MA, USA); voltage-dependent anion-selective channel protein 1 (VDAC1) antibody (catalog # PA1780) was purchased from Boster Biosciences (Pleasanton, CA, USA).

Techniques: Functional Assay, Knock-Out, Membrane

Journal: Autophagy

Article Title: Deregulated MTOR (mechanistic target of rapamycin kinase) is responsible for autophagy defects exacerbating kidney stone development

doi: 10.1080/15548627.2019.1635382

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Antibodies Product SQSTM1/p62 (sequestosome 1) MBL, PM045 phosphorylated SQSTM1/p62 (Ser 351; p-SQSTM1) MBL, PM074 LGALS3/galectin-3 (lectin, galactose binding, soluble 3) Santa Cruz Biotechnology, sc-23,938 mono- and polyubiquitinylated conjugated monoclonal antibody (FK2) BML, PW8810 LAMP1 (lysosomal-associated membrane protein 1) Abcam, ab24170 MAP1LC3 (microtubule-associated protein 1 light chain 3) MBL, PM036 TOMM20 (mitochondrial translocase of outer mitochondrial membrane 20) MBL, ab78547 PINK1 (PTEN induced putative kinase 1) Proteintech, 23,274–1-AP PRKN/PARK2/PARKIN (parkin RBR E3 ubiquitin protein ligase) Cell Signaling Technology, #2132 TFEB (transcription factor EB) Proteintech, 13,372–1-AP RPS6KB1/p70S6K Cell Signaling Technology, #9202 phosphorylated RPS6KB1/p70S6K (p-RPS6KB1/p70S6K) Cell Signaling Technology, #9234 ULK1 (unc-51 like kinase 1) Cell Signaling Technology, #8054 Phosphorylated ULK1 (p-ULK1) Cell Signaling Technology, #14,202 LRP2/MEGALIN (low density lipoprotein receptor-related protein 2) Abcam, ab76969 AQP2 (aquaporin 2) Abcam, ab199975 SLC12A3/thiazide-sensitive NaCl Cotransporter Merck KGaA, AB3553 COX4I1 (cytochrome c oxidase subunit 4I1) Proteintech, 11,242–1-AP 4ʹ,6-diamidino-2-phenylindole (DAPI) Dojindo, 340–07971 ACTB/β-actin Sigma-Aldrich, A5316 histone H3 Abcam, ab1791 Open in a separate window Antibodies used in this study.

Techniques: Binding Assay